Method of Identifying Membrane Ig Specific Antibodies and Use Thereof for Targeting Immunoglobulin-Producing Precursor Cells

ABSTRACT

The present invention relates to the discovery of antibodies that bind to novel epitopes present on membrane-anchored immunoglobulins and which bind to these novel epitopes on the surface of B cells and plasma cells. In addition, the antibodies of the present invention can mediate ADCC and can be useful to deplete those B cells and plasma cells expressing the novel epitopes of the invention. The antibodies of the present invention can be useful for the treatment of B cell-mediated diseases and diseases caused by monoclonal expansion of B cells. Accordingly the present invention also provides compositions and methods for the prevention, management, treatment or amelioration of B cell-mediated diseases and diseases caused by monoclonal expansion of B cells.

1. FIELD OF THE INVENTION

The present invention relates to agents that bind membrane-anchored IgEmolecules. In one embodiment, the present invention relates to agentsthat specifically bind to cells with membrane-anchored IgE and mediateantibody-dependent cellular cytotoxicity.

2. BACKGROUND OF THE INVENTION

IgE mediates, among other things, immediate-type hypersensitivityreactions. For an allergic reaction to occur, an individual must havehad prior exposure to an allergen. Following the initial antigenexposure, the immune system produces IgE specific for the incitingantigen. The antigen-specific IgE then binds to mast cell membranes viaIgE receptors. When re-exposed to the antigen, the antigen-specific IgEantibody binds to the antigen and activates the mast cells. Such mastcell activation causes a release of vasoactive and neuronal stimulatorymediators such as histamines, leukotrienes, prostaglandins, bradykinin,and platelet-activating factor which work in conjunction with cells suchas eosinophils, basophils, neutrophils, and CD4 T-lymphocytes. Allergeninduced IgE secretion can result in a variety of complications,including death, as may be the case in serious cases of asthma andanaphylaxis. These allergic disorders are prevalent. For example,allergic rhinitis (hay fever) affects 22% or more of the population ofthe USA, whereas allergic asthma is thought to affect at least 20million residents of the USA. The economic impact of allergic diseasesin the United States, including health care costs and lost productivity,has been estimated to amount to $6.4 billion in the early ninetiesalone.

IgE is secreted by IgE-producing plasma cells, which differentiate fromB cells expressing membrane-bound IgE (mIgE) on their surface. IgE notonly has the shortest biologic half-life of all classes ofimmunoglobulins (Igs), but also is present in serum at the lowestlevels. However, IgE concentrations in allergic reactions (atopic) inindividuals can be 100- to 1000-fold higher than in normal individuals.IgE is directly involved in mediating many allergic reactions as aresult of its ability to bind to and, upon contact with multivalentallergen, activate various effector cells, such as mast cells andbasophils, through interactions with FcεR1 receptors.

Since IgE plays a central role in mediating most allergic reactions,devising treatments to control IgE levels in the body and regulating IgEsynthesis has been of great interest. Several strategies have beenproposed to treat IgE-mediated allergic diseases by downregulating IgElevels. One strategy involves neutralizing the IgE molecules by bindingthe ε-chain of IgE in or near the Fc-receptor binding site. For example,Omalizumab (Xolair) is a recombinant humanized monoclonal anti-IgEantibody that binds to IgE on the same Fc site as FcεR1. Omalizumabcauses a reduction in total serum IgE in atopic patients, whichattenuates the amount of antigen-specific IgE that can bind to andsensitize tissue mast cells and basophils. This, in turn, leads to adecrease in symptoms of allergic diseases.

While Omalizumab reduces the amount of free IgE (the unbound formpresent in the circulation) it does not bind to IgE already bound toeffector cells nor does it bind to membrane-anchored IgE. Thus, whileneutralizing anti-IgE antibodies, like Omalizumab, may reduce theseverity of some IgE-mediated allergic diseases they may not beeffective for treating patients with very high levels of soluble IgE.Nor will they likely be effective for the treatment of diseases causedby monoclonal expansion of B-cells, such as, Job's disease. Strategiesto treat these diseases focus on depleting the B-cells producing IgE forexample, by binding membrane-anchored IgE present on the surface ofB-cells and targeting these cells for destruction by a variety ofmechanisms including the use of cytotoxic agents and mediating cellkilling pathways such as antibody dependent cell-mediated cytotoxicity(ADCC) and complement dependent cytotoxicity (CDC). These methods wouldbe efficacious for both the treatment of IgE-mediated allergic diseaseas well as for disease caused by the expansion of IgE expressingB-cells. Furthermore, these methods could be adapted to treat otherdiseases caused by monoclonal expansion of B-cells expressing othermembrane-anchored immunoglobulins such as, for example, IgM expressingB-cells in Waldenstrom Macroglubulinemia, IgA and IgG expressing B-cellsin various myelomas and autoimmune diseases and IgM and IgA expressingB-cells in neuropathy and nephropathy, post transplantlymphoproliferative disorder (PTLD), and monocolonal gammopathy ofunknown significance (MGUS).

There are two forms of immunoglobulins: the secreted and the membraneanchored form. The membrane-anchored form differs from the secreted formin that the former has a membrane-anchoring peptide extending from the Cterminus of the heavy-chain. Membrane-anchored immunoglobulin on B-cellsis critical for B-cell functions. It can transduce signals for resting Bcells to differentiate into activated lymphoblasts and Ig-secretingplasma cells. The amino acid sequences of many membrane-anchoredimmunoglobulins are known. These sequences share certain common featuresincluding the presence of a membrane anchoring peptide. The membraneanchoring peptide has three segments that are distinguishable based ontheir locations in relation to the plasma membrane (extracellularsegment, transmembrane segment, and cytoplasmic segment). The N-terminalsegment (extracellular segment) of the anchoring peptides is oftendesignated as hydrophilic and highly acidic. This segment can be easilyidentified by amino acid sequence comparison and analysis and isreferred to as the membrane-anchored immunoglobulin isotype specific(“migis”) peptide or epitope (see FIG. 1A).

The migis peptides are unique for the different immunoglobulin isotypes.Therefore, the extracellular segment of the ε-chain membrane anchoringpeptide forms, in whole or in part, an epitope unique to the B cellswhich produce IgE. The same is true for each immunoglobulin isotype.Furthermore, the migis peptide is not present on secreted, solubleimmunoglobulin because only the immunoglobulin which is bound to thesurface of B cells contains the membrane anchoring peptide as part ofits heavy chain. Thus, therapeutics which specifically targeted themigis peptides would be useful to target specific classes of B-cells forthe treatment of a wide variety of conditions including allergicdiseases and those mediated by monoclonal B-cell expansion.

Membrane anchored IgE is found in at least two isoforms as a result ofalternative splicing in humans. The ε-chain of both isoforms of humanmIgE contains a ε-migisepitope and a membrane-anchoring peptide. Oneisofolm contains only the s-migis sequence (a 15-amino-acid-long domain)between the membrane anchor sequence and the C4 region, referred to asthe short form. Whereas, the second isoform additionally contains anextra 52-amino-acid (a.a.)-long domain, referred to as cεmx, between theCH4 domain and the ε-migis sequence, referred to as the long form (seeFIG. 2). Several groups have generated mouse monoclonal antibodies thatbind to either the ε-migis peptide (see, e.g., Chang et al. U.S. Pat.Nos. 5,422,258 and 5,091,313) or an 8 amino acid cεmx peptide (Chen etal. 2002, Int Arch Allergy Immunol 128:315-24). However, as demonstratedherein (see Section 6, Example 1), antibodies that recognize the 8-migispeptide alone are likely to cross react with another commonly expressedcell surface protein, while those which interact with a predominantepitope present on the 8 amino acid cεmx peptide may in fact be hiddenwhen the immunoglobulin is present on the membrane. Furthermore, it isnot desirable to use mouse antibodies directly as a human therapeuticdue to the generation of human-anti-mouse antibodies (HAMA) or HAMAresponse. Thus, antibodies of non-human origin are preferably engineeredto “humanize” them to prevent eliciting a HAMA response. The process ofhumanization is not only time consuming but often results in an antibodywith altered binding characteristics that is not a useful therapeutic.The antibodies disclosed herein are fully human antibodies which bind aunique 8-chain migis epitope not previously described.

Citation or discussion of a reference herein shall not be construed asan admission that such is prior art to the present invention.

3. SUMMARY OF THE INVENTION

The present invention is based in part on the discovery of antibodiesthat specifically bind novel epitopes comprising at least a portion ofan e-migis peptide and a portion of the cεmx peptide, exemplified by SEQID NO: 5. The novel epitopes of the invention are referred to herein,for example, as “cεmx.migis epitope,” “cεmx.migis peptide,” or simply as“cεmx.migis,” and antigenic fragments thereof. The novel epitopes of theinvention are also encompassed, for example, by the more expansive terms“cεmx.migis epitopes of the invention,” or “cεmx.migis peptides of theinvention.” Antibodies that specifically bind novel cεmx.migis epitopesof the invention are specifically referred to herein as “cεmx.migisantibodies” and are also encompassed by the more expansive term“antibodies of the invention.” The present invention also providesmethods for the isolation of antibodies that bind novel epitopes andmethods of using the antibodies of the invention, for example, to treatIgE-mediated diseases.

Further, the present invention relates to the isolation of antibodieswhich specifically bind membrane-anchored Ig molecules (mIgs) includingbut not limited to membrane-anchored IgE molecules (mIgEs). In aspecific embodiment, the epitope recognized by antibodies whichspecifically bind mIgs include, but are not limited to, those describedherein (e.g., SEQ ID NO:5, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45and 46).

In another specific embodiment the antibodies which specifically bindmIgs including but not limited to mIgE (referred herein as antibodies ofthe invention) are human antibodies. In still another specificembodiment, antibodies of the invention mediate ADCC and/or CDCactivity.

In yet another specific embodiment, antibodies of the invention areuseful for the treatment of IgE-mediated disease and B-cell mediateddiseases including, but not limited to, asthma, allergic diseases, anddiseases caused by monoclonal expansion of B-cells such as, Job'sdisease.

4. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The Human Migis Peptides. Panel A provides the amino acidsequence of the 5 different human migis peptides the numbers inparenthesis indicate the percent homology of each peptide to that of IgEmigis. Panel B depicts the alignment of the ε-migis peptide with theμ-migis peptide. Solid lines indicate identity, dashed lines indicatedsimilarity. The μ-migis peptide share about 31% homology with 6-migis,with 4 identical residues and 5 similar residues over a 12 amino acidstretch. Also shown is the alignment of the e-migis peptide withpeptides from Phosphoinositide binding protein and from the predictedopen reading frame of KIAA1227, solid lines represent homology and thenon-homologous amino acids are indicated. 8 out of 11 amino acids areidentical between the phosphoinositide binding protein peptide and partof the s-migis peptide, while 8 out of 9 are identical between theKIAA1227 peptide and part of the s-migis peptide.

FIG. 2. Schematic of The ε-Chain Long And Short Splice Variants. Theopen box represents the variable region through the CH4 region, thehatched box represents the intracellular domain (ICD), the shaded boxrepresents the transmembrane domain (TM), the striped box represents thes-migis domain all of which are present in both the long and shortforms. The stippled box represents the cεmx region which is present onlyin the long form. The amino acid sequence of the cεmx.migis peptide usedfor panning is shown in black and grey indicating those residues derivedfrom the cεmx and ε-migis domains, respectively.

FIG. 3. Phage Binding Inhibition By The A1c Antibody Of Phage ClonesIsolated Via cεmx.migis Panning. The amount of binding seen for severalphage clones in the presence of an irrelevant isotype control antibodyis shown by the stippled boxes. The shaded boxes show the binding of thesame clone in the presence of the A1c antibody which is known to bind tothe “shared-epitope” of s-migis. The solid arrows indicate exemplaryclones which are inhibited by A1c while the open arrows are exemplaryclones which are not inhibited by A1c.

FIG. 4. Phage Binding Inhibition By The A1c and B1 Antibodies Of PhageClones Isolated Via cεmx.migis Panning. The amount of binding seen forseveral phage clones in the presence of an irrelevant isotype controlantibody is shown by the stippled boxes. The dark shaded boxes show thebinding of the same clone in the presence of the A1c antibody which ispresumed to bind to the “shared-epitope” of ε-migis. The light shadedboxes show the binding of the same clone in the presence of the B1antibody which is presumed to bind to an epitope hidden on mIgE(referred to as “hidden-epitope”) of cεmx.migis. The solid arrowsindicate clones which are inhibited by both A1c and B1 while the openarrows are clones which are not inhibited by either Ac1 or B1.

FIG. 5. FACS Analysis of Cell Surface Binding of Full Length IgGsGenerated From Non-Inhibited Phage Clones. The solid bars indicated thepercent (%) of cell staining while the shaded bars represent the meanchannel fluorescence (MCF). For a clone that specifically bound to thes-chain present only on the cells surface of transfected 293 cells(293-mIgE) both the percent of cells staining and the mean channelfluorescence should go down in untransfected 293 cells. Clone, D5(indicated by an arrow), shows such a staining pattern.

FIG. 6. FACS Analysis of Cell Surface Binding of D5 IgG. Panel A) Thesolid bars indicated the percent (%) of cell staining while the shadedbars represent the mean channel fluorescence (MCF). Panel B) Plotted isthe MCF of unstained cells (grey bars) and cells stained with D5 (darkbars) or a secondary antibody control (speckled bars), several differentcell types were examined as described below. Specific staining isindicated by staining (MCF value) with D5 but not by the secondarycontrol antibody. Together these data demonstrate that D5 stains cellswhich express membrane anchored IgE (239H-mIgE) but does notsignificantly stain cells expressing other immunoglobulins such as IgA(Daikiki cells) or IgM (RPMI 1788 cells) or cells not expressing anyimmunoglobulin such as untransfected 293 cells and CCRF-CEM (a T cellline) or RAJI cells. D5 was not seen to stain SKO-007 cells which havebeen reported to express mIgE but in a rather weak and unstable manner.

FIG. 7. ELISA Analysis of D5 IgG Binding. Panel A) ELISA binding assayshowing that D5 and B1 only bind to the cεmx.migis peptide while A1cbinds to an ε.migis peptide, a cεmx.migis peptide and a peptidecorresponding to the region of the phosphoionositide binding proteinthat is similar to s-migis (PIBP peptide) with nearly equal affinity.Panel B) ELISA binding assay of D5 IgG to Recombinant IgE, IgE.cεmx andIgE.cεmx.migis. The binding curves for serial dilutions of D5 IgG or acontrol antibody are represented by the solid and dotted linesrespectively. The diamonds indicate binding to rIgE, the squaresrepresent biding to rIgE.cεmx and the triangles represent binding torIgE.cεmx.migis. D5 IgG binds only rIgE.cεmx.migis, the control antibodydid not bind to any of the rIgE proteins. Panel C) ELISA Analysis of D5IgG Binding to Recombinant Full length IgG-Fc fused to either cεmx(IgG.Fc.cεmx) or cεmx.migis (IgG.Fc.cεmx.migis). Binding of D5 atconcentrations of 1.25 to 20 μg/ml to IgG.Fc.cεmx and IgG.Fc.cεmx.migisare shown by the dark and light bars, respectively. D5 IgG only binds tothe IgG.Fc.cεmx.migis molecule at each concentration examined.

FIG. 8. D5, F4 and D9 Selective Bind to The Cell Surface of CellsExpressing mIgE. The mean channel fluorescence is plotted for unstainedcells and cells stained either D5, F4, D9 and a secondary controlantibody. The D5, F4 and D9 antibodies each selectively stain only 293cells expressing mIgE, Igα and Igβ (grey bars) and not untransfected 293cells (black bars).

FIG. 9. BIAcore Analysis of D5 IgG Binding to the cεmx.migis and ε-migispeptides. The top trace represents the binding of the D5 IgG antibody tocεmx.migis while the bottom trace represents the binding of the D5antibody to ε-migis. D5 IgG only binds to cεmx.migis.

FIG. 10. ADCC activity of D5 IgG and the Fc-variant D53M as Measured byCell-based ADCC assay. Panel A are the results from donor 152. Panel Bare the results from donor 165. D5 IgG was seen to have higher ADCCactivity only in cells expressing IgE on their membranes (293-mIgE). Theassay was performed using 50:1 ratio of effector to target cells atantibody concentrations of 1 μg/ml and 10 μg/ml (shaded and dark barsrespectively). The Fc-variant D53M was seen to have more ADCC activitythen D5 IgG. Panel C are the results using three 293 cell line clones(1, 2 and 5) stably expressing mIgE, Igα and Igβ. D5 IgG has higher ADCCactivity against all three cell lines expressing mIgE as compared to acontrol IgG. The assay was performed using antibody concentrations of 1μg/ml, 10 μg/ml and 100 μg/ml (speckled, shaded and dark barsrespectively). D5 was seen to have as much as 70% cytotoxicity was seenfor the highest antibody concentrations as compared to ˜35% seen forcontrol antibodies. The control antibody does not show any difference inADCC activity between cells not expressing mIgE (293 cells) and thosethat do (clones 1, 2 and 5).

FIG. 11. ADCC activity of D5 IgG and the Fc-variant D53M IgG as Measuredby Cell-based ADCC assay. Both D5 IgG and the Fc variant, D53M IgG wereseen to specifically enhance ADCC in cells expressing IgE on theirmembranes. This activity could be specifically competed by the additionof the cεmx.migis peptide. In contrast, the activity of a controlFc-variant antibody did not depend on membrane expression of IgE. Threeantibody concentrations were used 1 μg/ml (shaded bars), 110 μg/ml(black bars) and 100 μg/ml (stippled bars). The Fc-variant D53M IgG wasseen to have more ADCC activity than D5 IgG.

FIG. 12. The nucleotide and deduced amino acid sequence of the variableregion of the D5 antibody (A) heavy chain variable region (SEQ ID NO: 8and SEQ ID NO: 10, respectively) (B) light chain variable region (SEQ IDNO: 7 and SEQ ID NO: 9, respectively). The CDRs are underlined (seeTable 1 for corresponding SEQ ID NOS.).

FIG. 13. The nucleotide and deduced amino acid sequence of the variableregion of the A1c antibody (A) heavy chain variable region (SEQ ID NO:51 and SEQ ID NO: 53, respectively) (B) light chain variable region (SEQID NO: 50 and SEQ ID NO: 52, respectively). The CDRs are underlined (seeTable 1 for corresponding SEQ ID NOS.).

FIG. 14. The nucleotide and deduced amino acid sequence of the variableregion of the B1 antibody (A) heavy chain variable region (SEQ ID NO: 61and SEQ ID NO: 63, respectively) (B) light chain variable region (SEQ IDNO: 60 and SEQ ID NO: 62, respectively). The CDRs are underlined (seeTable 1 for corresponding SEQ ID NOS.).

FIG. 15. The nucleotide and deduced amino acid sequence of the variableregion of the F4 antibody (A) heavy chain variable region (SEQ ID NO: 71and SEQ ID NO: 73, respectively) (B) light chain variable region (SEQ IDNO: 70 and SEQ ID NO: 72, respectively). The CDRs are underlined (seeTable 1 for corresponding SEQ ID NOS.).

FIG. 16. The nucleotide and deduced amino acid sequence of the variableregion of the D9 antibody (A) heavy chain variable region (SEQ ID NO: 80and SEQ ID NO: 81, respectively). The CDRs are underlined (see Table 1for corresponding SEQ ID NOS.).

FIG. 17. Stable Transfected 293 Cells Express mIgE, Igα and Igβ On TheirCell Surface. Plotted is the mean channel fluorescence of cell surfacestaining with anti-hu IgE, anti-Igα, anti-Igβ and a secondary antibodycontrol demonstrating that clones 1, 2 and 5 stain for all three cellsurface markers while control cells do not.

5. DETAILED DESCRIPTION

The present invention is based in part on the discovery of antibodiesthat specifically bind novel epitopes comprising at least a portion ofan 6-migis peptide and a portion of the cεmx peptide, exemplified by SEQID NO: 5. The novel epitopes of the invention are referred to herein,for example, as “cεmx.migis epitope,” “cεmx.migis peptide,” or simply as“cεmx.migis,” and antigenic fragments thereof. The novel epitopes of theinvention are also encompassed by the more expansive terms “migisepitopes of the invention,” and “migis epitopes.” Antibodies thatspecifically bind novel cεmx.migis epitopes of the invention arespecifically referred to herein as “cεmx.migis antibody(ies)” and arealso encompassed by the more expansive term “antibody(ies) of theinvention.” The present invention also provides methods for theisolation of antibodies that bind novel epitopes and methods of usingthe antibodies of the invention, for example, to treat IgE-mediateddiseases.

The cεmx.migis epitope to which the antibodies of the present inventionspecifically bind to is present on membrane anchored IgE (abbreviatedherein as “mIgE”). In one embodiment, the cεmx.migis antibodies thatspecifically bind the novel cεmx.migis epitope bind to mIgE. In anotherembodiment, antibodies of the invention which bind to mIgE mediate ADCCand/or CDC activity.

In one embodiment, an antibody of the invention specifically binds thepeptide sequence of human cεmx.migis peptide sequence (SEQ ID NO:5). Inanother embodiment, an antibody of the invention specifically binds tomembrane-anchored IgE (mIgE). In a specific embodiment, an antibody ofthe invention that specifically binds the peptide sequence of SEQ IDNO:5 does not bind membrane-anchored immunoglobulins (referred to hereinjointly as “mIgs”, and individually as “mIgG”, “mIgA”, “mIgE”, “mIgM”and “mIgD”) other than mIgE. In another specific embodiment, an antibodyof the invention that specifically binds the peptide sequence of SEQ IDNO:5 does not bind the peptide sequence of human ε-migis (SEQ ID NO: 1)and/or the human cεmx peptide sequence (SEQ ID NO:6). In still anotherspecific embodiment, an antibody of the invention that specificallybinds the peptide sequence of SEQ ID NO:5 does not bind the polypeptideof phosphoinositide binding protein epitope (SEQ ID NO:3) and/or theKIAA1227 peptide epitope (SEQ ID NO:4).

In one embodiment, an antibody of the invention that specifically bindsthe peptide sequence of SEQ ID NO:5 does not bind the same epitope as anantibody that specifically binds the peptide of SEQ ID NO: 1. In anotherembodiment, an antibody of the invention that specifically binds thepeptide sequence of SEQ ID NO:5 does not bind the same epitope as anantibody that specifically binds the peptide of SEQ ID NO:6. In stillanother embodiment, the binding of an antibody of the invention thatspecifically binds the peptide sequence of SEQ ID NO:5 to mIgE is notinhibited by the peptides of SEQ ID NO: 1 and SEQ ID NO:6.

In one embodiment, an antibody of the invention that specifically bindsthe peptide sequence of SEQ ID NO:5 does not bind the same epitope asantibodies comprising the variable regions of A1c (encoded by SEQ IDNOS: 50 and 51) and B1 (encoded by SEQ ID NOS: 60 and 61). In anotherembodiment, an antibody of the invention that specifically binds thepeptide sequence of SEQ ID NO:5 is not inhibited by antibodiescomprising the variable regions of A1c (encoded by SEQ ID NOS: 50 and51) and B1 (encoded by SEQ ID NOS: 60 and 61).

In one embodiment, an antibody of the invention that specifically bindsa migis epitope (e.g., a cεmx.migis epitope) depletes B cells or plasmacells expressing mIgE. Depletion may occur in an in vitro assay designedto measure B cell or plasma cell depletion, or may occur in vivo in asubject. In a more specific embodiment, an antibody of the inventionthat specifically binds the peptide sequence of SEQ ID NO:5 depletes Bcells or plasma cells expressing mIgE. In another specific embodiment,an antibody of the invention that specifically binds the peptidesequence of SEQ ID NO:5 depletes B cells or plasma cells expressing mIgEthrough ADCC. In still another specific embodiment, an antibody of theinvention that specifically binds the peptide sequence of SEQ ID NO:5depletes B cells or plasma cells expressing mIgE through CDC.

In one embodiment, an antibody of the invention specifically binds apeptide having an amino acid sequence selected from the group consistingof SEQ ID NO:5, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46.

Antibodies or fragments that specifically bind to a peptide (e.g.,csmx.migis) can be identified, for example, by immunoassays, BIAcore, orother techniques known to those of skill in the art. An antibody orfragment thereof binds specifically to a migis epitope or a fragmentthereof when it binds to a migis epitope or a fragment thereof withhigher affinity than to any cross-reactive antigen as determined usingexperimental techniques, such as radioimmunoassays (RIA) andenzyme-linked immunosorbent assays (ELISAs). See, e.g., Paul, ed., 1989,Fundamental Immunology Second Edition, Raven Press, New York at pages332-336 for a discussion regarding antibody specificity.

The present invention further encompasses antibodies of the inventionthat have a high binding affinity for a migis epitope (e.g., acεmx.migis epitope). In a specific embodiment, an antibody of theinvention that specifically binds to a migis epitope has an associationrate constant or k_(on) rate of at least 10⁵M⁻¹s⁻¹, at least 5×10⁵M⁻¹s⁻,at least 10⁶M⁻¹s⁻¹, at least 5×10⁶M⁻¹s⁻¹, at least 10⁷M⁻¹s⁻¹, at least5×10⁷M⁻¹s⁻¹, or at least 10⁸M⁻¹s⁻¹. In another embodiment, an antibodyof the invention that specifically binds to a migis epitope has a k_(on)of at least 2×10⁵M⁻¹s⁻¹, at least 5×10⁵M⁻¹s⁻¹, at least 10⁶M⁻¹s⁻¹, atleast 5×10⁶M⁻¹s⁻¹, at least 10⁷M⁻¹s⁻¹, at least 5×10⁷M⁻¹s⁻¹, or at least10⁸M⁻¹s⁻¹. In a particular embodiment, the migis epitope for which anantibody of the invention has a k_(on) as disclosed herein is acεmx.migis epitope. In another particular embodiment, the migis epitopefor which an antibody of the invention has a k_(on) as disclosed hereinis a peptide selected from the group consisting of SEQ ID NO:5, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45 and 46. In a specific embodiment, themigis epitope for which an antibody of the invention has a k_(on) asdisclosed herein is a peptide having the amino acid sequence of SEQ IDNO:5.

In another embodiment, an antibody of the invention that specificallybinds to a migis epitope has a k_(off) of less than 10⁻¹s⁻¹, less than5×10⁻¹s⁻¹, less than 10⁻²s⁻¹, less than 5×10⁻² s⁻¹, less than 10⁻³s⁻¹,less than 5×10⁻³s⁻¹, less than 10⁻⁴s⁻¹, less than 5×10⁻⁴s⁻¹, less than10⁻⁵s⁻¹, less than 5×10⁻⁵s⁻¹, less than 10⁻⁶s⁻¹, less than 5×10⁻⁶s⁻¹,less than 10⁻⁷s⁻¹, less than 5×10⁻⁷s⁻¹, less than 10⁻⁸s⁻¹, less than5×10⁹s⁻¹, less than 10⁻⁹s⁻¹, less than 5×10⁻⁹s⁻¹, or less than 10⁻⁸s⁻¹.In another embodiment, an antibody of the invention that specificallybinds to a migis epitope has a k_(off), of less than 5×10⁻⁴s⁻¹, lessthan 10⁻⁵s⁻¹, less than 5×10⁻⁵s⁻¹, less than 10⁻⁶s⁻¹, less than5×10⁻⁶s⁻¹, less than 10⁻⁷s⁻¹, less than 5×10⁻⁷s⁻¹, less than 10⁻⁸s⁻¹,less than 5×10⁻⁸s⁻¹, less than 10⁻⁹s⁻¹, less than 5×10⁻⁹s⁻¹, or lessthan 1⁻¹⁰s⁻¹. In a particular embodiment, the migis epitope for which anantibody of the invention has a k_(off) as disclosed herein is acεmx.migis epitope. In another particular embodiment, the migis epitopefor which an antibody of the invention has a k_(off) as disclosed hereinis a peptide selected from the group consisting of SEQ ID NO:5, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45 and 46. In a specific embodiment, themigis epitope for which an antibody of the invention has a k_(off) asdisclosed herein is a peptide having the amino acid sequence of SEQ IDNO:5.

In another embodiment, an antibody of the invention that specificallybinds to a migis epitope has an affinity constant or K_(a)(k_(on)/k_(off)) of at least 10²M⁻¹, at least 5×10²M⁻¹, at least 10³M⁻¹,at least 5×10³M⁻¹, at least 10⁴M⁻¹, at least 5×10⁴M⁻¹, at least 10⁵M⁻¹,at least 5×10⁵M⁻¹, at least 10⁶M⁻¹, at least 5×10⁶M⁻¹, at least 10⁷M⁻¹,at least 5×10⁷M⁻¹, at least 10⁸M⁻¹, at least 5×10⁸M⁻¹, at least 10⁹M⁻¹,at least 5×10⁹M⁻¹, at least 10¹⁰M⁻¹, at least 5×10¹⁰M⁻¹, at least10¹¹M⁻¹, at least 5×10¹¹M⁻¹, at least 10¹²M⁻¹, at least 5×10¹²M, atleast 10¹³M⁻¹, at least 5×10¹³M⁻¹, at least 10¹⁴M⁻¹, at least 5×10¹⁴M⁻¹,at least 10¹⁵M⁻¹, or at least 5×10¹⁵M⁻¹. In a particular embodiment, themigis epitope for which an antibody of the invention has a k_(a) asdisclosed herein is a cεmx.migis epitope. In another particularembodiment, the migis epitope for which an antibody of the invention hasa k_(a) as disclosed herein is a peptide selected from the groupconsisting of SEQ ID NO:5, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45and 46. In a specific embodiment, the migis epitope for which anantibody of the invention has a k_(a) as disclosed herein is a peptidehaving the amino acid sequence of SEQ ID NO:5.

In yet another embodiment, an antibody of the invention thatspecifically binds to a migis epitope has a dissociation constant orK_(d) (k_(off)/k_(on)) of less than 10⁻²M, less than 5×10⁻²M, less than10⁻³M, less than 5×10⁻³M, less than 10⁻⁴M, less than 5×10⁻⁴M, less than10⁻⁵M, less than 5×10⁻⁵M, less than 10⁻⁶M, less than 5×10⁻⁶M, less than10⁻⁷M, less than 5×10⁻⁷M, less than 10⁻⁸M, less than 5×10⁻⁸M, less than10⁹M, less than 5×10⁻⁹M, less than 10⁻¹⁰M, less than 5×10⁻¹⁰M, less than10⁻¹¹M, less than 5×10⁻¹¹M, less than 10⁻¹²M, less than 5×10⁻¹²M, lessthan 10⁻¹³M, less than 5×10⁻¹³M, less than 10⁻¹⁴M, less than 5×10⁻¹⁴M,less than 10⁻¹⁵M, or less than 5×10⁻¹⁵M. In still another embodiment, anantibody of the invention that specifically binds to a migis epitope hasa dissociation constant or K_(d) (k_(off)/k_(on)) of between about 10⁻⁷Mand about 10⁻⁸M, between about 10⁻⁸M and about 10⁻⁹M, between about10⁻⁹M and about 10⁻¹⁰M, between about 10⁻¹⁰M and about 10⁻¹⁰M, betweenabout 10⁻¹⁰M and about 10⁻¹²M, between about 10⁻¹²M and about 10⁻¹³M,between about 10⁻¹³M and about 10⁻¹⁴M. In still another embodiment, anantibody of the invention that specifically binds to a migis epitope hasa dissociation constant or K_(d) (k_(off)/k_(on)) of between 10⁻⁷M and10⁻⁸M, between 10⁻⁸M and 10⁻⁹M, between 10⁻⁹M and 10⁻¹⁰M, between 10⁻¹⁰Mand 10⁻¹¹M, between 10⁻¹¹M and 10⁻¹²M, between 10⁻¹²M and 10⁻¹³M,between 10¹³M and 10⁻¹⁴M. In a particular embodiment, the migis epitopefor which an antibody of the invention has a k_(d) as disclosed hereinis a cεmx.migis epitope. In another particular embodiment, the migisepitope for which an antibody of the invention has a k_(d) as disclosedherein is a peptide selected from the group consisting of SEQ ID NO:5,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 and 46. In a specificembodiment, the migis epitope for which an antibody of the invention hasa k_(d) as disclosed herein is a peptide having the amino acid sequenceof SEQ ID NO:5.

It is well known in the art that the equilibrium dissociation constant(K_(d)) is defined as k_(off)/k_(on). It is generally understood that abinding molecule (e.g., and antibody) with a low K_(d) is preferable toa binding molecule (e.g., and antibody) with a high K_(d). However, insome instances the value of the k_(on) or k_(off) may be more relevantthan the value of the K_(d). One skilled in the art can determine whichkinetic parameter is most important for a given antibody application. Incertain embodiments, the antibodies of the invention have a lower K_(d)for one antigen than for others.

In one embodiment, an antibody of the invention has at least 2, at least5, at least 10, at least 10², at least 5×10², at least 10³, at least5×10³, at least 10⁴, at least 5×10⁴, at least 10⁵, at least 5×10⁵, or atleast 10⁶ fold lower K_(d) for a peptide having the amino acid sequenceof SEQ ID NO:5 compared to the K_(d) for a peptide having the amino acidsequence of SEQ ID NO:1.

In one embodiment, an antibody of the invention has at least 2, at least5, at least 10, at least 10², at least 5×10², at least 10³, at least5×10³, at least 10⁴, at least 5×10⁴, at least 10⁵, at least 5×10⁵, or atleast 10⁶ fold lower K_(d) for a peptide having the amino acid sequenceof SEQ ID NO:5 compared to the Kd for a peptide having the amino acidsequence of SEQ ID NO:6.

In one embodiment, an antibody of the invention has at least 2, at least5, at least 10, at least 10², at least 5×10², at least 10³, at least5×10³, at least 10⁴, at least 5×10⁴, at least 10⁵, at least 5×10⁵, or atleast 10⁶ fold lower I(d for a peptide having the amino acid sequence ofSEQ ID NO:5 compared to the Kd for a peptide having the amino acidsequence of SEQ ID NO: 1 and a peptide having the amino acid sequence ofSEQ ID NO:6.

The present invention comprises antibodies that specifically bind to apeptide having the amino acid sequence of SEQ ID NO:5. The presentinvention also provides antibodies that specifically bind tomembrane-anchored IgE (mIgE). In certain embodiments, an antibody of theinvention binds to a peptide having the amino acid sequence of SEQ IDNO:5 and binds to mIgE. In one embodiment an antibody of the inventionthat specifically bind to a peptide having the amino acid sequence ofSEQ ID NO:5 and/or specifically binds to mIgE comprises a variable lightchain (V_(L)) domain of SEQ ID NO:9. In a particular embodiment, anantibody of the invention that specifically bind to a peptide having theamino acid sequence of SEQ ID NO:5 and/or specifically binds to mIgEcomprises a V_(L) domain encoded by the nucleotide of SEQ ID NO: 7. Inanother embodiment, an antibody of the invention that specifically bindto a peptide having the amino acid sequence of SEQ ID NO:5 and/orspecifically binds to mIgE comprises a variable heavy chain (V_(H))domain of SEQ ID NO: 10. In a particular embodiment, an antibody of theinvention that specifically bind to a peptide having the amino acidsequence of SEQ ID NO:5 and/or specifically binds to mIgE comprises aV_(H) domain encoded by the nucleotide of SEQ ID NO: 8. In a specificembodiment, an antibody of the invention that specifically bind to apeptide having the amino acid sequence of SEQ ID NO:5 and/orspecifically binds to mIgE comprises a V_(L) domain of SEQ ID NO:9 andV_(H) domain of SEQ ID NO:10. In a particular embodiment, an antibody ofthe invention that specifically bind to a peptide having the amino acidsequence of SEQ ID NO:5 and/or specifically binds to mIgE comprises aV_(L) domain encoded by the nucleotide of SEQ ID NO: 7 and a V_(H)domain encoded by the nucleotide of SEQ ID NO: 8.

In one embodiment an antibody of the invention that specifically bind toa peptide having the amino acid sequence of SEQ ID NO:5 and/orspecifically binds to mIgE comprises a variable light chain (V_(L))domain of SEQ ID NO:60. In a particular embodiment, an antibody of theinvention that specifically bind to a peptide having the amino acidsequence of SEQ ID NO:5 and/or specifically binds to mIgE comprises aV_(L) domain encoded by the nucleotide of SEQ ID NO: 62. In anotherembodiment, an antibody of the invention that specifically bind to apeptide having the amino acid sequence of SEQ ID NO:5 and/orspecifically binds to mIgE comprises a variable heavy chain (V_(H))domain of SEQ ID NO:63. In a particular embodiment, an antibody of theinvention that specifically bind to a peptide having the amino acidsequence of SEQ ID NO:5 and/or specifically binds to mIgE comprises aV_(H) domain encoded by the nucleotide of SEQ ID NO: 61. In a specificembodiment, an antibody of the invention that specifically bind to apeptide having the amino acid sequence of SEQ ID NO:5 and/orspecifically binds to mIgE comprises a V_(L) domain of SEQ ID NO:62 andV_(H) domain of SEQ ID NO:63. In a particular embodiment, an antibody ofthe invention that specifically bind to a peptide having the amino acidsequence of SEQ ID NO:5 and/or specifically binds to mIgE comprises aV_(L) domain encoded by the nucleotide of SEQ ID NO: 60 and a V_(H)domain encoded by the nucleotide of SEQ ID NO: 61.

The present invention comprises antibodies that specifically binds tomembrane-anchored IgE (mIgE). In one embodiment an antibody of theinvention that specifically bind to that specifically binds tomembrane-anchored IgE (mIgE) comprises a variable heavy chain (V_(H))domain of SEQ ID NO:81. In a particular embodiment, an antibody of theinvention that specifically bind to a peptide having the amino acidsequence of SEQ ID NO:5 comprises a V_(H) domain encoded by thenucleotide of SEQ ID NO: 80.

In other embodiments, an antibody of the invention that specificallybind to a peptide having the amino acid sequence of SEQ ID NO:5 and/orspecifically binds to mIgE comprises at least one, at least 2, at least3, at least 4, at least 5, or at least 6 CDRs from the antibody D5 orF4. In still other embodiments, an antibody of the invention thatspecifically binds to mIgE comprises at least one, at least 2, at least3 CDRs from the antibody D9. The amino acid sequence of the CDRs ofantibody D5 are indicated in FIG. 12 and are represented by SEQ ID NOS:11 (V_(L) CDR1), 12 (V_(L) CDR2), 13 (V_(L) CDR3), 14 (V_(H) CDR1),(V_(H) CDR2), and 16 (V_(H) CDR3). The amino acid sequence of the CDRsof antibody F4 are indicated in FIG. 15 and are represented by SEQ IDNOS: 74 (V_(L) CDR1), 75 (V_(L) CDR2), 76 (V_(L) CDR3), 77 (V_(H) CDR1),78 (V_(H) CDR2), and 79 (V_(H) CDR3). The amino acid sequence of theCDRs of antibody D9 are indicated in FIG. 16 and are represented by SEQID NOS: 82 (V_(H) CDR1), 83 (V_(H) CDR2), and 84 (V_(H) CDR3).

The present invention also encompasses antibodies that specifically bindto a peptide having the amino acid sequence of SEQ ID NO:5 and/orspecifically binds to mIgE comprising at least one CDR selected from thegroup comprising: a CDR that is at least 80% identical to the lightchain CDR1 of the D5 antibody, a CDR that is at least 80% identical tothe light chain CDR2 of the D5 antibody, a CDR that is at least 80%identical to the light chain CDR3 of the D5 antibody, a CDR that is atleast 80% identical to the heavy chain CDR1 of the D5 antibody, a CDRthat is at least 80% identical to the heavy chain CDR2 of the D5antibody, a CDR that is at least 80% identical to the heavy chain CDR3of the D5 antibody, a CDR that is at least 80% identical to the lightchain CDR1 of the F4 antibody, a CDR that is at least 80% identical tothe light chain CDR2 of the F4 antibody, a CDR that is at least 80%identical to the light chain CDR3 of the F4 antibody, a CDR that is atleast 80% identical to the heavy chain CDR1 of the F4 antibody, a CDRthat is at least 80% identical to the heavy chain CDR2 of the F4antibody, a CDR that is at least 80% identical to the heavy chain CDR3of the F4 antibody, a CDR that is at least 80% identical to the heavychain CDR1 of the D9 antibody, a CDR that is at least 80% identical tothe heavy chain CDR2 of the D9 antibody, and a CDR that is at least 80%identical to the heavy chain CDR3 of the D9 antibody. Also contemplatedare antibodies that specifically bind to a peptide having the amino acidsequence of SEQ ID NO:5 and/or specifically binds to mIgE having atleast one CDR that is at least 80%, or at least 85%, or at least 90%, orat least 95%, or at least 98%, or at least 99% identical to a CDRpresent in an antibody selected from the group consisting of D5, F4 andD9.

The present invention further encompasses nucleotides encodingantibodies of the invention. In one embodiment, an isolated nucleic acidsequence of the invention encodes the amino acid sequence of SEQ ID NO:9or 10. In a specific embodiment, an isolated nucleic acid sequence ofthe invention comprises SEQ ID NO: 7 or 8. In another embodiment, anisolated nucleic acid sequence of the invention encodes the amino acidsequence of SEQ ID NO:72 or 73. In a specific embodiment, an isolatednucleic acid sequence of the invention comprises SEQ ID NO: 70 or 71. Instill another embodiment, an isolated nucleic acid sequence of theinvention encodes the amino acid sequence of SEQ ID NO:81. In a specificembodiment, an isolated nucleic acid sequence of the invention comprisesSEQ ID NO: 80. Also encompassed by the present invention are cellcomprising at least a nucleic acid sequence of the invention encodingthe amino acid sequence of SEQ ID NO:9, 10, 72, 73 or 81.

In another embodiment, an isolated nucleic acid sequence of theinvention encodes a polypeptide that is at least 60% identical, or atleast 70% identical, or at least 80% identical, or at least 85%identical, or at least 90% identical, or at least 95% identical, or atleast at least 97% identical, or at least 99% identical, or 100%identical to the amino acid sequence of SEQ ID NO:9, 10, 72, 73 or 81.In still another embodiment, an isolated nucleic acid sequence of theinvention is at least 60% identical, or at least 70% identical, or atleast 80% identical, or at least 85% identical, or at least 90%identical, or at least 95% identical, or at least at least 97%identical, or at least 99% identical, or 100% identical to the nucleicacid sequence of SEQ ID NO:7, 8, 70, 71 or 80.

The percent identity of two amino acid sequences (or two nucleic acidsequences) can be determined, for example, by aligning the sequences foroptimal comparison purposes (e.g., gaps can be introduced in thesequence of a first sequence). The amino acids or nucleotides atcorresponding positions are then compared, and the percent identitybetween the two sequences is a function of the number of identicalpositions shared by the sequences (i.e., % identity=# of identicalpositions/total # of positions×100). The actual comparison of the twosequences can be accomplished by well-known methods, for example, usinga mathematical algorithm. A preferred, non-limiting example of such amathematical algorithm is described in Karlin et al., Proc. Natl. Acad.Sci. USA, 90:5873-5877 (1993). Such an algorithm is incorporated intothe BLASTN and BLASTX programs (version 2.2) as described in Schaffer etal., Nucleic Acids Res., 29:2994-3005 (2001). When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., BLASTN) can be used. See http://www.ncbi.nlm.nih.gov, asavailable on Apr. 10, 2002. In one embodiment, the database searched isa non-redundant (NR) database, and parameters for sequence comparisoncan be set at: no filters; Expect value of 10; Word Size of 3; theMatrix is BLOSUM62; and Gap Costs have an Existence of 11 and anExtension of 1.

Another preferred, non-limiting example of a mathematical algorithmutilized for the comparison of sequences is the algorithm of Myers andMiller, CABIOS (1989). Such an algorithm is incorporated into the ALIGNprogram (version 2.0), which is part of the GCG (Accelrys) sequencealignment software package. When utilizing the ALIGN program forcomparing amino acid sequences, a PAM120 weight residue table, a gaplength penalty of 12, and a gap penalty of 4 can be used. Additionalalgorithms for sequence analysis are known in the art and includeADVANCE and ADAM as described in Torellis and Robotti, Comput. Appl.Biosci., 10: 3-5 (1994); and FASTA described in Pearson and Lipman,Proc. Natl. Acad. Sci. USA, 85: 2444-8 (1988).

In another embodiment, the percent identity between two amino acidsequences can be accomplished using the GAP program in the GCG softwarepackage (available at http://www.accelrys.com, as available on Aug. 31,2001) using either a Blossom 63 matrix or a PAM250 matrix, and a gapweight of 12, 10, 8, 6, or 4 and a length weight of 2, 3, or 4. In yetanother embodiment, the percent identity between two nucleic acidsequences can be accomplished using the GAP program in the GCG softwarepackage (available at http://www.cgc.com), using a gap weight of 50 anda length weight of 3.

It is contemplated that the present invention also encompassesantibodies that bind the same epitope as an antibody comprising thevariable regions of D5 (encoded by SEQ ID NO: 7 and 8). Also encompassedare antibodies that compete for binding of the epitope of an antibodycomprising the variable regions of D5 (encoded by SEQ ID NO: 7 and 8).The present invention further encompasses antibodies that bind the sameepitope as an antibody comprising the variable regions of D5 (encoded bySEQ ID NO: 7 and 8) that have a K_(d) between about 10⁻⁷M and about10⁻⁸M, between about 10⁻⁸M and about 10⁻⁹M, between about 10⁻⁹M andabout 10⁻¹⁰M, between about 10⁻¹⁰M and about 10⁻¹¹M, between about10⁻¹¹M and about 10⁻¹²M, between about 10⁻¹²M and about 10⁻¹³M, betweenabout 10⁻¹³M and about 10⁻¹⁴M.

As disclosed herein (see, e.g., Example 3) the antibodies of theinvention can mediate ADCC against target cells expressing mIg, inparticular mIgE. Thus, the cεmx.migis antibodies disclosed herein areuseful for the treatment of IgE-mediated disorders, including but notlimited to, those resulting from or associated with the binding of IgEto FcεRI and those caused by monoclonal expansion of IgE expressingB-cells. An IgE mediated or associated disease or disorder includes, forexample, allergic disease caused by IgE antibodies and mast cellmediators including but not limited to atopic diseases such as allergicrhinitis, allergic asthma, including asthma associated with specificantigenic factors such as seasonal pollens (grass: rye, timothy,ragweed) and tree (birch), perennial allergens such as dust mite, animaldanders, feathers and fungal spores and occupational antigens such asdetergents and metals as well as asthma associated with non-antigenspecific factors such as infection, irritants such as smoke, fumes,diesel exhaust particles and sulphur dioxide, asthma associated withairway cooling (exercise, cold air temperatures) and emotional stress;atopic dermatitis and allergic gastroenteropathy as well as anaphylacticdiseases including systemic anaphylaxis and reactions to proteins infoods (e.g., peanuts), venom, vaccines, hormones, antiserum, enzymes andlatex, reactions to haptens including antibiotics, muscle relaxants,vitamins, cytotoxins and opiates and reactions to polysaccharides suchas dextran, iron dextran and polygeline and other anaphylactic diseasesor disorders such as urticaria-angioedema, as well as B-cell expansiondiseases such as Job's disease, post transplant lymphoproliferativedisorder (PTLD), and monocolonal gammopath of unknown significance(MGUS). In addition, the antibodies of the invention are useful for thetreatment of IgE-mediated gastro-intestinal inflammatory disorders whichcan be broadly defined as intractable chronic responses to a broad rangeof host reaction to a variety of insults, such as those caused by injuryor infection which are characterized by, or results from pathologyaffected by IgE. Particular disorders included within the scope of theterm includes inflammatory bowel disease (e.g., Crohn's disease,ulcerative colitis, indeterminate colitis and infectious colitis),gastroenteropathy, enteritis, mucositis (e.g., oral mucositis,gastrointestinal mucositis, nasal mucositis and proctitis), necrotizingenterocolitis and esophagitis).

Accordingly, the present invention provides methods useful for theprevention, management, treatment or amelioration of B-cell mediateddiseases and disorders including, those resulting from or associatedwith monoclonal expansion of B-cells, and in particular those mediatedby IgE. In one embodiment, the invention provides methods of treating anIgE-mediated disease in a human comprising administering to anindividual in need of such prevention, amelioration, or treatment aneffective amount of an antibody of the invention.

In a specific embodiment, the invention provides methods of treating anIgE-mediated disease in a human comprising administering to anindividual in need of such prevention, amelioration, or treatment aneffective amount of antibody of the invention that specifically binds toa peptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:5, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45and 46.

In another specific embodiment, the invention provides methods oftreating an IgE-mediated disease in a human comprising administering toan individual in need of such prevention, amelioration, or treatment aneffective amount of antibody of the invention in combination with otherprophylactic or therapeutic agents.

As described above, the migis peptides, which are present only onmembrane-anchored immunoglobulins, are unique for the differentimmunoglobulin isotypes. Therefore, this extracellular segment of theimmunoglobulin membrane anchoring peptide forms, in whole or in part, anepitope unique to the B cells expressing a particular membrane-anchoredimmunoglobulin isotype. Thus, therapeutics, such as antibodies, whichspecifically target the migis peptides would be useful to targetspecific classes of B-cells for the treatment of a wide variety ofconditions including allergic diseases and those mediated by monoclonalB-cell expansion.

However, as disclosed herein, when identifying antibodies which are bothspecific for a particular migis peptide and which bind membrane-anchoredimmunoglobulin, one must account for the presence of predominantepitopes within the migis peptides which may have undesirablecharacteristics. For example, as demonstrated herein, a predominantmigis epitope may be shared with other proteins or may be hidden on themembrane-anchored immunoglobulin. Accordingly, the present inventionalso provides methods for identification, isolation and use ofantibodies which do not bind to predominant epitopes present on anypolypeptide of interest (e.g., migis peptides, and peptides comprisingmigis peptides and fragments thereof).

The present invention encompasses a method of producing an antibody thatdoes not bind to a predominant epitope comprising: (a) screening anantibody library before or after selection for antibodies which bind toa polypeptide comprising a predominant epitope for antibodies which arenot inhibited by an antibody recognizing the predominant epitope presenton said polypeptide; and (b) isolating at least one antibody from (a).It is contemplated that a predominant epitope is one which isaccessible, antigenic and furthermore is an epitope to which antibodiesare readily generated, identified, or isolated. A single polypeptide maycomprise more than one predominant epitope. A predominant epitope may bea linear polypeptide sequence or may result from the three dimensionalconfirmation of a polypeptide. It is further contemplated that apredominant epitope is one which will be recognized by multiple antibodybinding domains present in an antibody library. Antibodies recognizing apredominant epitope may be antibodies previously isolated from the samelibrary as that used in the methods of the present invention. Anantibody may be considered to bind a predominant epitope if, forexample, it competes for binding with other antibodies which bind thesame polypeptide. In one embodiment, a method of producing an antibodythat does not bind to a predominant epitope of a polypeptide comprisinga migis peptide (or fragment thereof) is provided herein. For example, amethod of producing such antibodies to a mIgE peptide is providedherein.

In one embodiment, the method of producing an antibody that does notbind to a predominant epitope comprises: (a) isolating from an antibodylibrary those clones which bind to a polypeptide comprising thepredominant epitope; (b) screening the clones isolated in (a) for thosewhich are not inhibited by an antibody that specifically binds thepredominant epitope; and (c) isolating at least one antibody from (b).In one embodiment, a method of producing an antibody that does not bindto a predominant epitope of a polypeptide comprising a migis peptide (orfragment thereof) is provided herein. For example, a method of producingsuch antibodies to a mIgE peptide is provided herein.

Methods for generating and isolating clones from antibody libraries arewell known in the art. Some representative methods are disclosed inSection 5.3 entitled “Methods of Generating Antibodies” and in Section6.1, Example 1. Methods to screen antibodies for those which do or don'tinhibit the binding of another antibody are well known in the art.Several representative methods are disclosed in section 5.5 entitled“Biological Assays” and in Section 6.1, Example 1. The antibody clonesidentified by the method of the invention may be readily isolated bymethods well known in the art. It is contemplated that antibody clonescould be isolated from an antibody library in the presence of one ormore antibodies which bind predominant epitopes present on thepolypeptide comprising the predominant epitope thereby isolating onlythose antibody clones which are not inhibited by one or more antibodieswhich bind predominant epitopes. The use of antibodies which bindpredominant epitopes during the isolation of antibody clones from anantibody library may reduce the number of clones to be screened foradditional desired binding properties (e.g., specificity for mIg).

The methods of the invention may also be useful for the production ofantibodies that specifically bind a migis epitope which is not apredominant epitope, is not shared by other proteins and which is nothidden on a membrane-anchored immunoglobulin. In one embodiment, themethod for producing antibodies that specifically bind a migis epitopewhich is not a predominant epitope, is not shared by other proteins andwhich is not hidden on a membrane-anchored immunoglobulin comprises: (a)isolating from an antibody library those clones which bind to apolypeptide comprising the migis epitope; (b) screening the clonesisolated from step (a) for those which are not inhibited by an antibodyrecognizing the predominant epitope present on said polypeptidecomprising the migis epitope; (c) screening the clones which are notinhibited in step (b) for those which specifically bind cells having themembrane anchored immunoglobulin; (d) screening the clones whichspecifically bound in step (c) for those which do not bind cells nothaving the membrane anchored immunoglobulin; and (e) isolating at leastone antibody from (d).

Accordingly, the methods of the present invention are useful for theproduction of antibodies which specifically bind membrane-anchoredimmunoglobulins (referred to herein jointly as “mIgs”, and individuallyas “mIgG”, “mIgA”, “mIgE”, “mIgM” and “mIgD”), do not bind predominantepitopes and do not bind other proteins. In a specific embodiment, theepitope recognized by antibodies which specifically bind mIgs is a migisepitope. Exemplary migis epitopes include, but are not limited to, thoseshown in FIG. 1A and those described herein as SEQ ID NOS.: 1, 2, 5, 47,48 and 49. Other exemplary migis epitopes include amino acid residuescomprising a migis peptide and one or more amino acid residues of theadjacent heavy chain sequences (e.g., SEQ ID NO:5 and 17 to 49).

In one embodiment, antibodies produced by the methods of the inventionspecifically bind mIgs. In a specific embodiment, antibodies produced bythe methods of the invention are human antibodies. In still anotherspecific embodiment, antibodies produced by the methods of the inventionmediate ADCC and/or CDC activity. In yet another specific embodiment,antibodies produced by the methods of the invention are useful for thetreatment of B-cell mediated diseases including but not limited toasthma, allergic diseases (e.g., by targeting B-cell expressing mIgE),myelomas, autoimmune and inflammatory diseases such as rheumatoidarthritis, and lupus (e.g., by targeting B-cells expressing mIgG ormIgA), and diseases caused by monoclonal expansion of B-cells such as,Job's disease (e.g., by targeting B-cell expressing mIgE), Waldenstrommacroglubulinemia (e.g., by targeting B-cell expressing mIgM), posttransplant lymphoproliferative disorder (PTLD), and monocolonalgammopath of unknown significance (MGUS).

In one specific embodiment, antibodies produced by the methods of theinvention specifically bind a migis epitope present on the α-chain. Inanother specific embodiment, antibodies produced by the methods of theinvention specifically bind a migis epitope present on the δ-chain. Instill another specific embodiment, antibodies produced by the methods ofthe invention specifically bind a migis epitope present on the γ-chain.In yet another specific embodiment, antibodies produced by the methodsof the invention specifically bind a migis epitope present on theμ-chain. In still another specific embodiment, antibodies produced bythe methods of the invention specifically bind a migis epitope presenton the ε-chain.

In one embodiment, antibodies produced by the methods of the inventionspecifically bind to membrane anchored immunoglobulins (mIgs). Inanother embodiment, antibodies produced by the methods of the inventionspecifically bind to mIgA but do not bind mIgs other than mIgA. In stillanother embodiment, antibodies produced by the methods of the inventionspecifically bind to mIgD but do not bind mIgs other than mIgD. In stillanother embodiment, antibodies produced by the methods of the inventionspecifically bind to mIgG but do not bind mIgs other than mIgG. In yetanother embodiment, antibodies produced by the methods of the inventionspecifically bind to mIgM but do not bind mIgs other than mIgM. In stillanother embodiment, antibodies produced by the methods of the inventionspecifically bind to mIgE but do not bind mIgs other than mIgE.

In one embodiment, an antibody produced by the methods of the inventionwhich specifically binds a migis epitope depletes B cells or plasmacells expressing mIg. Depletion may occur in an in vitro assay designedto measure B cell or plasma cell depletion, or may occur in vivo in asubject. In a specific embodiment, an antibody produced by the methodsof the invention which specifically binds a migis epitope depletes Bcells or plasma cells expressing mIg through ADCC. In another specificembodiment, an antibody produced by the methods of the invention whichspecifically binds a migis epitope depletes B cells or plasma cellsexpressing mIg through CDC.

In one embodiment, an antibody produced by the methods of the inventionwhich specifically binds a migis epitope depletes B cells or plasmacells expressing mIgA. In a specific embodiment, an antibody produced bythe methods of the invention which specifically binds a migis epitopedepletes B cells or plasma cells expressing mIgD. In another specificembodiment, an antibody produced by the methods of the invention whichspecifically binds a migis epitope depletes B cells or plasma cellsexpressing mIgG. In yet another specific embodiment, an antibodyproduced by the methods of the invention which specifically binds amigis epitope depletes B cells or plasma cells expressing mIgM.

5.1 Antibodies of the Invention

As used herein, the terms “antibody” and “antibodies” refer tomonoclonal antibodies, multispecific antibodies, human antibodies,humanized antibodies, camelised antibodies, chimeric antibodies,single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), Fab fragments, F(ab′) fragments, and anti-idiotypic (anti-Id) antibodies (including,e.g., anti-Id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. In particular, antibodiesinclude immunoglobulin molecules and immunologically active fragments ofimmunoglobulin molecules, i.e., molecules that contain an antigenbinding site, these fragments may or may not be fused to anotherimmunoglobulin domain including but not limited to, an Fc region orfragment thereof. As outlined herein, the terms “antibody” and“antibodies” specifically include the cεmx.migis antibodies describedherein, full length antibodies and Fc variants thereof comprising Fcregions, or fragments thereof, comprising at least one novel amino acidresidue described herein fused to an immunologically active fragment ofan immunoglobulin or to other proteins as described herein. Such valiantFc fusions include but are not limited to, scFv-Fc fusions, variableregion (e.g., VL and VH)-Fc fusions, scFv-scFv-Fc fusions.Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD,IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) orsubclass.

Antibodies of the invention may include, but are not limited to,synthetic antibodies, monoclonal antibodies, oligoclonal antibodies,recombinantly produced antibodies, intrabodies, multispecificantibodies, bispecific antibodies, human antibodies, humanizedantibodies, chimeric antibodies, synthetic antibodies, single-chainFv-Fc fusions (scv-Fc), single-chain Fv (scFv), and anti-idiotypic(anti-Id) antibodies. In particular, antibodies used in the methods ofthe present invention include immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules. Theantibodies of the invention can be of any type (e.g., IgG, IgE, IgM,IgD, IgA and IgY), class (e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA, and IgA₂)or subclass of immunoglobulin molecule.

Antibodies of the invention may be from any animal origin includingbirds and mammals (e.g., human, rodent, donkey, sheep, rabbit, goat,guinea pig, camel, horse, or chicken). In one embodiment, the antibodiesare human or humanized monoclonal antibodies. As used herein, “human”antibodies include antibodies having the amino acid sequence of a humanimmunoglobulin and include antibodies isolated from human immunoglobulinlibraries or from mice that express antibodies from human genes.Antibodies like all polypeptides have an Isoelectric Point (pI), whichis generally defined as the pH at which a polypeptide carries no netcharge. It is known in the art that protein solubility is typicallylowest when the pH of the solution is equal to the isoelectric point(pI) of the protein. It is possible to optimize solubility by alteringthe number and location of ionizable residues in the antibody to adjustthe pI. For example the pI of a polypeptide can be manipulated by makingthe appropriate amino acid substitutions (e.g., by substituting acharged amino acid such as a lysine, for an uncharged residue such asalanine). Without wishing to be bound by any particular theory, aminoacid substitutions of an antibody that result in changes of the pI ofsaid antibody may improve solubility and/or the stability of theantibody. One skilled in the art would understand which amino acidsubstitutions would be most appropriate for a particular antibody toachieve a desired pI. The pI of a protein may be determined by a varietyof methods including but not limited to, isoelectric focusing andvarious computer algorithms (see for example Bjellqvist et al., 1993,Electrophoresis 14:1023-1031). In one embodiment, the pI of theantibodies of the invention is higher than about 6.5, about 7.0, about7.5, about 8.0, about 8.5, or about 9.0. In one embodiment,substitutions resulting in alterations in the pI of the antibody of theinvention will not significantly diminish its binding affinity for amigis epitope. In another embodiment, the pI of the antibodies of theinvention is higher then 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0. It isspecifically contemplated that the substitution(s) of the Fc region thatresult in altered binding to FcγR (described supra) may also result in achange in the pI. In another embodiment, substitution(s) of the Fcregion are specifically chosen to effect both the desired alteration inFcγR binding and any desired change in pI. As used herein the pI valueis defined as the pI of the predominant charge form. The pI of a proteinmay be determined by a variety of methods including but not limited to,isoelectric focusing and various computer algorithms (see, e.g.,Bjellqvist et al., 1993, Electrophoresis 14:1023).

The thermal melting temperatures (Tm) of the Fab domain of an antibody,can be a good indicator of the thermal stability of an antibody and mayfurther provide an indication of the shelf-life. A lower Tm indicatesmore aggregation/less stability, whereas a higher Tm indicates lessaggregation/more stability. Thus, antibodies having higher Tm arepreferable. In one embodiment, the Fab domain of an antibody of theinvention has a Tm value higher than at least 50° C., 55° C., 60° C.,65° C., 70° C., 75° C., 80° C., 85° C., 90° C., 95° C., 100° C., 105°C., 110° C., 115° C. or 120° C. Tm of a protein domain (e.g., a Fabdomain) can be measured using any standard method known in the art, forexample, by differential scanning calorimetry (see, e.g., Vermeer etal., 2000, Biophys. J. 78:394-404; Vermeer et al., 2000, Biophys. J. 79:2150-2154).

Antibodies of the invention may be monospecific, bispecific, trispecificor have greater multispecificity. Multispecific antibodies mayimmunospecifically bind to different epitopes of desired target moleculeor may immunospecifically bind to both the target molecule as well as aheterologous epitope, such as a heterologous polypeptide or solidsupport material. See, e.g., International Publication Nos. WO 94/04690;WO 93/17715; WO 92/08802; WO 91/00360; and WO 92/05793; Tutt, et al.,1991, J. Immunol. 147:60-69; U.S. Pat. Nos. 4,474,893, 4,714,681,4,925,648, 5,573,920, and 5,601,819; and Kostelny et al., 1992, J.Immunol. 148:1547-1553). In the present case, one of the bindingspecificities is for a migis epitope (e.g., cεmx.migis) and the otherone is for any other antigen (e.g., CD3, a signaling or effectormolecule).

Multispecific antibodies have binding specificities for at least twodifferent antigens. While such molecules normally will only bind twoantigens (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by theinstant invention. Examples of BsAbs include without limitation thosewith one arm directed against a migis epitope and the other arm directedagainst any other antigen. Methods for making bispecific antibodies areknown in the art. Traditional production of full-length bispecificantibodies is based on the coexpression of two immunoglobulin heavychain-light chain pairs, where the two chains have differentspecificities (Millstein et al., 1983, Nature, 305:537-539). Because ofthe random assortment of immunoglobulin heavy and light chains, thesehybridomas (quadromas) produce a potential mixture of different antibodymolecules, of which only one has the correct bispecific structure.Purification of the correct molecule, which is usually done by affinitychromatography steps, is rather cumbersome, and the product yields arelow. Similar procedures are disclosed in WO 93/08829, and in Trauneckeret al., 1991, EMBO J., 10:3655-3659. A more directed approach is thegeneration of a Di-diabody a tetravalent bispecific antibody. Methodsfor producing a Di-diabody are known in the art (see e.g., Lu et al.,2003, J Immunol Methods 279:219-32; Marvin et al., 2005, ActaPharmacolical Sinica 26:649).

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is preferred to havethe first heavy-chain constant region (CH1) containing the sitenecessary for light chain binding, present in at least one of thefusions. DNAs encoding the immunoglobulin heavy chain fusions and, ifdesired, the immunoglobulin light chain, are inserted into separateexpression vectors, and are co-transfected into a suitable hostorganism. This provides for great flexibility in adjusting the mutualproportions of the three polypeptide fragments in embodiments whenunequal ratios of the three polypeptide chains used in the constructionprovide the optimum yields. It is, however, possible to insert thecoding sequences for two or all three polypeptide chains in oneexpression vector when, the expression of at least two polypeptidechains in equal ratios results in high yields or when the ratios are ofno particular significance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm (e.g., a migis epitope such as csmx.migis), and ahybrid immunoglobulin heavy chain-light chain pair (providing a secondbinding specificity) in the other arm. It was found that this asymmetricstructure facilitates the separation of the desired bispecific compoundfrom unwanted immunoglobulin chain combinations, as the presence of animmunoglobulin light chain in only one half of the bispecific moleculeprovides for a facile way of separation. This approach is disclosed inWO 94/04690. For further details of generating bispecific antibodiessee, for example, Suresh et al., 1986, Methods in Enzymology, 121:210.According to another approach described in WO96/27011, a pair ofantibody molecules can be engineered to maximize the percentage ofheterodimers which are recovered from recombinant cell culture. Thepreferred interface comprises at least a part of the CH3 domain of anantibody constant domain. In this method, one or more small amino acidside chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089) Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Antibodies with more than two valencies incorporating at least one hingemodification of the invention are contemplated. For example, trispecificantibodies can be prepared. See, e.g., Tutt et al. J. Immunol. 147: 60(1991).

The antibodies of the invention encompass single domain antibodies,including camelized single domain antibodies (see e.g., Muyldermans etal., 2001, Trends Biochem. Sci. 26:230; Nuttall et al., 2000, Cur.Pharm. Biotech. 1:253; Reichmann and Muyldermans, 1999, J. Immunol.Meth. 231:25; International Publication Nos. WO 94/04678 and WO94/25591; U.S. Pat. No. 6,005,079.

Other antibodies specifically contemplated are “oligoclonal” antibodies.As used herein, the term “oligoclonal” antibodies” refers to apredetermined mixture of distinct monoclonal antibodies. See, e.g., PCTpublication WO 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163. In oneembodiment, oligoclonal antibodies consist of a predetermined mixture ofantibodies against one or more epitopes are generated in a single cell.In another embodiment, oligoclonal antibodies comprise a plurality ofheavy chains capable of pairing with a common light chain to generateantibodies with multiple specificities (e.g., PCT publication WO04/009618). Oligoclonal antibodies are particularly useful when it isdesired to target multiple epitopes on a single target molecule (e.g.,mIgE, mIgG, mIgA, mIgD, mIgM). Those skilled in the art will know or candetermine what type of antibody or mixture of antibodies is applicablefor an intended purpose and desired need.

Antibodies of the present invention also encompass antibodies that havehalf-lives (e.g., serum half-lives) in a mammal, (e.g., a human), ofgreater than 5 days, greater than 10 days, greater than 15 days, greaterthan 20 days, greater than 25 days, greater than 30 days, greater than35 days, greater than 40 days, greater than 45 days, greater than 2months, greater than 3 months, greater than 4 months, or greater than 5months. The increased half-lives of the antibodies of the presentinvention in a mammal, (e.g., a human), results in a higher serum titerof said antibodies or antibody fragments in the mammal, and thus,reduces the frequency of the administration of said antibodies orantibody fragments and/or reduces the concentration of said antibodiesor antibody fragments to be administered. Antibodies having increased invivo half-lives can be generated by techniques known to those of skillin the art. For example, antibodies with increased in vivo half-livescan be generated by modifying (e.g., substituting, deleting or adding)amino acid residues identified as involved in the interaction betweenthe Fc domain and the FcRn receptor (see, e.g., InternationalPublication Nos. WO 97/34631; WO 04/029207; U.S. Pat. No. 6,737,056 andU.S. Patent Publication No. 2003/0190311).

In one embodiment, the antibodies of the invention may be chemicallymodified (e.g., one or more chemical moieties can be attached to theantibody) or be modified to alter its glycosylation, again to alter oneor more functional properties of the antibody.

In still another embodiment, the glycosylation of the antibodies of theinvention is modified. For example, an aglycoslated antibody can be made(i.e., the antibody lacks glycosylation). Glycosylation can be alteredto, for example, increase the affinity of the antibody for a targetantigen. Such carbohydrate modifications can be accomplished by, forexample, altering one or more sites of glycosylation within the antibodysequence. For example, one or more amino acid substitutions can be madethat result in elimination of one or more variable region frameworkglycosylation sites to thereby eliminate glycosylation at that site.Such aglycosylation may increase the affinity of the antibody forantigen. Such an approach is described in further detail in U.S. Pat.Nos. 5,714,350 and 6,350,861.

Additionally or alternatively, an antibody of the invention can be madethat has an altered type of glycosylation, such as a hypofucosylatedantibody having reduced amounts of fucosyl residues or an antibodyhaving increased bisecting GlcNAc structures. Such altered glycosylationpatterns have been demonstrated to increase the ADCC ability ofantibodies. Such carbohydrate modifications can be accomplished by, forexample, expressing the antibody in a host cell with alteredglycosylation machinery. Cells with altered glycosylation machinery havebeen described in the art and can be used as host cells in which toexpress recombinant antibodies of the invention to thereby produce anantibody with altered glycosylation. See, for example, Shields, R. L. etal. (2002) J. Biol. Chem. 277:26733-26740; Umnana et al (1999) Nat.Biotech. 17:176-1, as well as, European Patent No: EP 1,176,195; PCTPublications WO 03/035835; WO 99/54342.

In still another embodiment, the glycosylation of an antibody of theinvention is modified. For example, an aglycoslated antibody can be made(i.e., the antibody lacks glycosylation). Glycosylation can be alteredto, for example, increase the affinity of the antibody for a targetantigen. Such carbohydrate modifications can be accomplished by, forexample, altering one or more sites of glycosylation within the antibodysequence. For example, one or more amino acid substitutions can be madethat result in elimination of one or more variable region frameworkglycosylation sites to thereby eliminate glycosylation at that site.Such aglycosylation may increase the affinity of the antibody forantigen. Such an approach is described in further detail in U.S. Pat.Nos. 5,714,350 and 6,350,861.

Additionally or alternatively, an antibody of the invention can be madethat has an altered type of glycosylation, such as a hypofucosylatedantibody of the invention having reduced amounts of fucosyl residues oran antibody of the invention having increased bisecting GlcNAcstructures. Such altered glycosylation patterns have been demonstratedto increase the ADCC ability of antibodies. Such carbohydratemodifications can be accomplished by, for example, expressing theantibody in a host cell with altered glycosylation machinery. Cells withaltered glycosylation machinery have been described in the art and canbe used as host cells in which to express recombinant antibodies of theinvention to thereby produce an antibody with altered glycosylation.See, for example, Shields, R. L. et al. (2002) J. Biol. Chem.277:26733-26740; Umana et al (1999) Nat. Biotech. 17:176-1, as well as,European Patent No: EP 1,176,195; PCT Publications WO 03/035835; WO99/54342.

5.2 Antibody Conjugates and Derivatives

Antibodies of the invention include derivatives that are modified (i.e.,by the covalent attachment of any type of molecule to the antibody suchthat covalent attachment). For example, but not by way of limitation,the antibody derivatives include antibodies that have been modified,e.g., by glycosylation, acetylation, pegylation, phosphorylation,amidation, derivatization by known protecting/blocking groups,proteolytic cleavage, linkage to a cellular ligand or other protein,etc. Any of numerous chemical modifications may be carried out by knowntechniques, including, but not limited to, specific chemical cleavage,acetylation, formylation, metabolic synthesis of tunicamnycin, etc.Additionally, the derivative may contain one or more non-classical aminoacids.

Antibodies or fragments thereof with increased in vivo half-lives can begenerated by attaching to said antibodies or antibody fragments polymermolecules such as high molecular weight polyethyleneglycol (PEG). PEGcan be attached to said antibodies or antibody fragments with or withouta multifunctional linker either through site-specific conjugation of thePEG to the N- or C-terminus of said antibodies or antibody fragments orvia epsilon-amino groups present on lysine residues. Linear or branchedpolymer derivatization that results in minimal loss of biologicalactivity will be used. The degree of conjugation will be closelymonitored by SDS-PAGE and mass spectrometry to ensure proper conjugationof PEG molecules to the antibodies. Unreacted PEG can be separated fromantibody-PEG conjugates by, e.g., size exclusion or ion-exchangechromatography.

Further, antibodies can be conjugated to albumin in order to make theantibody or antibody fragment more stable in vivo or have a longer halflife in vivo. The techniques are well known in the art, see e.g.,International Publication Nos. WO 93/15199, WO 93/15200, and WO01/77137; and European Patent No. EP 413, 622. The present inventionencompasses the use of antibodies or fragments thereof conjugated orfused to one or more moieties, including but not limited to, peptides,polypeptides, proteins, fusion proteins, nucleic acid molecules, smallmolecules, mimetic agents, synthetic drugs, inorganic molecules, andorganic molecules.

The present invention encompasses the use of antibodies or fragmentsthereof recombinantly fused or chemically conjugated (including bothcovalent and non-covalent conjugations) to a heterologous protein orpolypeptide (or fragment thereof, preferably to a polypeptide of atleast 10, at least 20, at least 30, at least 40, at least 50, at least60, at least 70, at least 80, at least 90 or at least 100 amino acids)to generate fusion proteins. The fusion does not necessarily need to bedirect, but may occur through linker sequences. For example, antibodiesmay be used to target heterologous polypeptides to particular celltypes, either in vitro or in vivo, by fusing or conjugating theantibodies to antibodies specific for particular cell surface receptors.Antibodies fused or conjugated to heterologous polypeptides may also beused in in vitro immunoassays and purification methods using methodsknown in the art. See e.g., International publication No. WO 93/21232;European Patent No. EP 439,095; Naramura et al., 1994, Immunol. Lett.39:91-99; U.S. Pat. No. 5,474,981; Gillies et al, 1992, PNAS89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452.

The present invention further includes formulations comprisingheterologous proteins, peptides or polypeptides fused or conjugated toantibody fragments. For example, the heterologous polypeptides may befused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)₂fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragmentthereof. Methods for fusing or conjugating polypeptides to antibodyportions are well known in the art. See, e.g., U.S. Pat. Nos. 5,336,603,5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; EuropeanPatent Nos. EP 307,434 and EP 367,166; International publication Nos. WO96/04388 and WO 91/06570; Ashblenazi et al., 1991, Proc. Natt. Acad.Sci. USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600;and Vil et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337-11341.

Additional fusion proteins, e.g., of anti-migis antibodies, may begenerated through the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”). DNA shuffling may be employed to alter the activities ofantibodies of the invention or fragments thereof (e.g., antibodies orfragments thereof with higher affinities and lower dissociation rates).See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238; 5,830,721;5,834,252; and 5,837,458, and Patten et al., 1997, Curr. OpinionBiotechnol. 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2): 76-82;Hansson, et al., 1999, J. Mol. Biol. 287:265-76; and Lorenzo and Blasco,1998, Biotechniques 24(2): 308-313. Antibodies or fragments thereof, orthe encoded antibodies or fragments thereof, may be altered by beingsubjected to random mutagenesis by error-prone PCR, random nucleotideinsertion or other methods prior to recombination. One or more portionsof a polynucleotide encoding an antibody or antibody fragment, whichportions specifically bind to a migis epitope may be recombined with oneor more components, motifs, sections, parts, domains, fragments, etc. ofone or more heterologous molecules.

Moreover, the antibodies or fragments thereof can be fused to markersequences, such as a peptide to facilitate purification. In specificembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., 1989, Proc. Natl.Acad. Sci. USA 86:821-824, for instance, hexa-histidine provides forconvenient purification of the fusion protein. Other peptide tags usefulfor purification include, but are not limited to, the hemagglutinin “HA”tag, which corresponds to an epitope derived from the influenzahemagglutinin protein (Wilson et al., 1984, Cell 37:767) and the “flag”tag.

In other embodiments, antibodies of the invention or analogs orderivatives thereof are conjugated to a diagnostic or detectable agent.Such antibodies can be useful for monitoring or prognosing thedevelopment or progression of a cancer as part of a clinical testingprocedure, such as determining the efficacy of a particular therapy.Such diagnosis and detection can be accomplished by coupling theantibody to detectable substances including, but not limited to variousenzymes, such as but not limited to horseradish peroxidase, alkalinephosphatase, beta-galactosidase, or acetylcholinesterase; prostheticgroups, such as but not limited to streptavidin/biotin andavidin/biotin; fluorescent materials, such as but not limited to,umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;luminescent materials, such as but not limited to, luminol;bioluminescent materials, such as but not limited to, luciferase,luciferin, and aequorin; radioactive materials, such as but not limitedto iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S), tritium(³H), indium (¹¹⁵In, ¹¹³In, ¹¹²In, ¹¹¹In,), and technetium (⁹⁹Tc),thallium (201Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum(⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm,¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴² Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and¹¹⁷Tin; positron emitting metals using various positron emissiontomographies, noradioactive paramagnetic metal ions, and molecules thatare radiolabelled or conjugated to specific radioisotopes.

The present invention further encompasses uses of antibodies of theinvention or fragments thereof conjugated to a therapeutic agent.

In other embodiments, antibodies of the invention may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters. A cytotoxin or cytotoxic agent includes any agent thatis detrimental to cells. Examples include paclitaxel, cytochalasin B,gramicidin D, ethidium bromide, emetine, mitomycin, etoposide,tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, puromycin, epirubicin, andcyclophosphamide and analogs or homologs thereof. Therapeutic agentsinclude, but are not limited to, antimetabolites (e.g., methotrexate,6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracildecarbazine), alkylating agents (e.g., mechlorethamine, thioepachlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines(e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics(e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, andanthramycin (AMC)), anti-mitotic agents (e.g., vincristine andvinblastine), and auristatin E compounds (e.g. monomethyl auristatin E;see for example U.S. Pat. No. 6,884,869). A more extensive list oftherapeutic moieties can be found in PCT publications WO 03/075957;

In other embodiments, antibodies of the invention may be conjugated to atherapeutic agent or drug moiety that modifies a given biologicalresponse. Therapeutic agents or drug moieties are not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, Onconase (or another cytotoxic RNase), pseudomonas exotoxin,cholera toxin, or diphtheria toxin; a protein such as tumor necrosisfactor, α-interferon, β-interferon, nerve growth factor, plateletderived growth factor, tissue plasminogen activator, an apoptotic agent,e.g., TNF-α, TNF-β, AIM I (see, International Publication No. WO97/33899), AIM II (see, International Publication No. WO 97/34911), FasLigand (Takahashi et al., 1994, J. Immunol., 6:1567), and VEGI (see,International Publication No. WO 99/23105), a thrombotic agent or ananti-angiogenic agent, e.g., angiostatin or endostatin; or, a biologicalresponse modifier such as, for example, a lymphokine (e.g.,interleukin-1 (“IL-1”), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”),granulocyte macrophage colony stimulating factor (“GM-CSF”), andgranulocyte colony stimulating factor (“G-CSF”)), or a growth factor(e.g., growth hormone (“GH”)).

In other embodiments, antibodies of the invention can be conjugated totherapeutic moieties such as a radioactive materials or macrocyclicchelators useful for conjugating radiometal ions (see above for examplesof radioactive materials). In certain embodiments, the macrocyclicchelator is 1,4,7,10-tetraazacyclododecane-N,N′,N″,N″-tetraacetic acid(DOTA) which can be attached to the antibody via a linker molecule. Suchlinker molecules are commonly known in the art and described in Denardoet al., 1998, Clin Cancer Res. 4:2483-90; Peterson et al., 1999,Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol.26:943-50.

Techniques for conjugating therapeutic moieties to antibodies are wellknown. Moieties can be conjugated to antibodies by any method known inthe art, including, but not limited to aldehyde/Schiff linkage,sulphydryl linkage, acid-labile linkage, cis-aconityl linkage, hydrazonelinkage, enzymatically degradable linkage (see generally Gamett, 2002,Adv Drug Deliv Rev 53:171-216). Techniques for conjugating therapeuticmoieties to antibodies are well known, see, e.g., Amon et al.,“Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy”,in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp.243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., “Antibodies For DrugDelivery”, in Controlled Drug Delivery (2nd Ed.), Robinson et al.(eds.), pp. 623-53 (Marcel Delker, Inc. 1987); Thorpe, “AntibodyCarriers Of Cytotoxic Agents In Cancer Therapy: A Review”, in MonoclonalAntibodies '84: Biological And Clinical Applications, Pinchera et al.(eds.), pp. 475-506 (1985); “Analysis, Results, And Future ProspectiveOf The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., 1982,Immunol. Rev. 62:119-58.

Methods for fusing or conjugating antibodies to polypeptide moieties areknown in the art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929;5,359,046; 5,349,053; 5,447,851, and 5,112,946; EP 307,434; EP 367,166;PCT Publications WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991,PNAS USA 88:10535-10539; Zheng et al., 1995, J Immunol 154:5590-5600;and Vil et al., 1992, PNAS USA 89:11337-11341. The fusion of an antibodyto a moiety does not necessarily need to be direct, but may occurthrough linker sequences. Such linker molecules are commonly known inthe art and described in Denardo et al., 1998, Clin Cancer Res4:2483-90; Peterson et al., 1999, Bioconjug Chem 10:553; Zimmerman etal., 1999, Nucl Med Biol 26:943-50; Garnett, 2002, Adv Drug Deliv Rev53:171-216.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

The therapeutic moiety or drug conjugated to an antibody or fragmentthereof that specifically binds to a migis epitope should be chosen toachieve the desired prophylactic or therapeutic effect(s) for aparticular disorder in a subject. A clinician or other medical personnelshould consider the following when deciding on which therapeutic moietyor drug to conjugate to an antibody or fragment thereof thatspecifically binds to a migis epitope: the nature of the disease, theseverity of the disease, and the condition of the subject.

5.3 Methods of Generating Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or by recombinant expression techniques.

Polyclonal antibodies to a migis epitope (e.g., cεmx.migis) can beproduced by various procedures well known in the art. For example, amigis epitope (e.g., cεmx.migis) or immunogenic fragments thereof can beadministered to various host animals including, but not limited to,rabbits, mice, rats, etc. to induce the production of sera containingpolyclonal antibodies specific for migis epitope (e.g., cεmx.migis).Various adjuvants may be used to increase the immunological response,depending on the host species, and include but are not limited to,Freund's (complete and incomplete), mineral gels such as aluminumhydroxide, surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanins, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and corynebacterium parvum. Suchadjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981). The term “monoclonalantibody” as used herein is not limited to antibodies produced throughhybridoma technology. The term “monoclonal antibody” refers to anantibody that is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. Briefly,mice can be immunized with migis epitope (e.g., cεmx.migis) or afragment thereof and once an immune response is detected, e.g.,antibodies specific for migis epitope are detected in the mouse serum,the mouse spleen is harvested and splenocytes isolated. The splenocytesare then fused by well known techniques to any suitable myeloma cells,for example cells from cell line SP20 available from the ATCC.Additionally, a RIMMS (repetitive immunization, multiple sites)technique can be used to immunize an animal (Kilpatrick et al., 1997,Hybridoma 16:381-9). Hybridomas are selected and cloned by limiteddilution. The hybridoma clones are then assayed by methods known in theart for cells that secrete antibodies capable of binding a polypeptideof the invention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, monoclonal antibodies can be generated by culturing ahybridoma cell secreting an antibody wherein, preferably, the hybridomais generated by fusing splenocytes isolated from a mouse immunized withmigis epitope (e.g., cεmx.migis) or immunogenic fragments thereof, withmyeloma cells and then screening the hybridomas resulting from thefusion for hybridoma clones that secrete an antibody able to bind amigis epitiope, and more specifically, a migis epitope in the context ofa membrane-anchored immunoglobulin molecule,

The antibodies of the invention contain novel amino acid residues intheir Fc regions. antibodies can be generated by numerous methods wellknown to one skilled in the art. Non-limiting examples include,isolating antibody coding regions (e.g., from hybridoma) and making oneor more desired substitutions in the Fc region of the isolated antibodycoding region. Alternatively, the variable regions may be subcloned intoa vector encoding an Fc region comprising one or more high effectorfunction amino acid residues. Additional methods and details areprovided below.

Antibody fragments that recognize specific migis epitopes may begenerated by any technique known to those of skill in the art. Forexample, Fab and F(ab′)2 fragments of the invention may be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)2fragments). F(ab′)2 fragments contain the variable region, the lightchain constant region and the CH1 domain of the heavy chain. Further,the antibodies of the present invention can also be generated usingvarious phage display methods known in the art.

In one embodiment, antibodies that specifically bind a migis epitope maybe generated by phage display methods.

In phage display methods, functional antibody domains are displayed onthe surface of phage particles that carry the polynucleotide sequencesencoding them. In particular, DNA sequences encoding VH and VL domainsare amplified from animal cDNA libraries (e.g., human or murine cDNAlibraries of lymphoid tissues). The DNA encoding the VH and VL domainsare recombined together with an scFv linker by PCR and cloned into aphagemid vector (e.g., p CANTAB 6 or pComb 3 HSS). The vector iselectroporated in E. coli and the E. coli is infected with helper phage.Phage used in these methods are typically filamentous phage including fdand M13 and the VH and VL domains are usually recombinantly fused toeither the phage gene III or gene VIII. Phage expressing an antigenbinding domain that binds to the migis epitope of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Examples of phagedisplay methods that can be used to make the antibodies of the presentinvention include those disclosed in Brinkman et al., 1995, J. Immunol.Methods 182:41-50; Ames et al., 1995, J. Immunol. Methods 184:177-186;Kettleborough et al., 1994, Eur. J. Immunol. 24:952-958; Persic et al.,1997, Gene 187:9-18; Burton et al., 1994, Advances in Immunology57:191-280; PCT Publication Nos. WO 90/02809, WO 91/10737, WO 92/01047,WO 92/18619, WO 93/11236, WO 95/15982, WO 95/20401, and WO97/13844; andU.S. Pat. Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908,5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225,5,658,727, 5,733,743 and 5,969,108

In a specific embodiment, an antibody specifically binding a migisepitope is produced by screening a library for antibodies that bind SEQID NO:5 and bind with at least 2, at least 5, at least 10-fold lessaffinity to SEQ ID NO:1 and SEQ ID NO:6. In another specific embodiment,an antibody specifically binding a migis epitope is produced byscreening a library for antibodies that that bind SEQ ID NO:5 andwherein the binding to SEQ ID NO:5 is not inhibited by an antibodiescomprising the variable regions of A1c (encoded by SEQ ID NOS: 50 and51) and B1 (encoded by SEQ ID NOS: 60 and 61).

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described below. Techniques to recombinantly produceFab, Fab′ and F(ab′)2 fragments can also be employed using methods knownin the art such as those disclosed in International Publication No. WO92/22324; Mullinax et al., 1992, BioTechniques 12(6): 864-869; Sawai etal., 1995, AJRI 34:26-34; and Better et al., 1988, Science240:1041-1043.

To generate whole antibodies, PCR primers including a portion of the VHor VL nucleotide sequences, a restriction site, and a flanking sequenceto facilitate digestion of the restriction site can be used to amplifythe VH or VL sequences in scFv clones. Utilizing cloning techniquesknown to those of skill in the art, the PCR amplified VH domains can becloned into vectors expressing a VH constant region, e.g., the humangamma constant, and the PCR amplified VL domains can be cloned intovectors expressing a VL constant region, e.g., human kappa or lambaconstant regions. In one embodiment, the constant region is an Fc regioncontaining at least one high effector function amino acid. In a specificembodiment, the vectors for expressing the VH or VL domains comprise apromoter, a secretion signal, a cloning site for both the variable andconstant domains, as well as a selection marker such as neomycin. The VHand VL domains may also be cloned into one vector expressing the desiredconstant regions. The heavy chain conversion vectors and light chainconversion vectors are then co-transfected into cell lines to generatestable or transient cell lines that express full-length antibodies,e.g., IgG, using techniques known to those of skill in the art.

In one embodiment, the antibodies of the invention are chimericantibodies. A chimeric antibody is a molecule in which differentportions of the antibody are derived from different immunoglobulinmolecules. Methods for producing chimeric antibodies are known in theart. See e.g., Morrison, 1985, Science 229:1202; Oi et al., 1986,BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods125:191-202; and U.S. Pat. Nos. 5,807,715, 4,816,567, 4,816,397, and6,311,415.

For some uses, including in vivo use of antibodies in humans and invitro detection assays, it may be preferable to use human or chimericantibodies. Completely human antibodies are particularly desirable formost therapeutic treatments of human subjects. Human antibodies can bemade by a variety of methods known in the art including phage displaymethods described above using antibody libraries derived from humanimmunoglobulin sequences. See also U.S. Pat. Nos. 4,444,887 and4,716,111; and PCT Publication Nos. WO 98/46645, WO 98/50433, WO98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741. In oneembodiment, the antibodies of the invention are human antibodies.

A humanized antibody is an antibody or its variant or fragment thereofwhich is capable of binding to a predetermined antigen and whichcomprises a framework region having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains (Fab, Fab′, F(ab′)₂, Fabc, Fv) in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin(i.e., donor antibody) and all or substantially all of the frameworkregions are those of a human immunoglobulin consensus sequence. In oneembodiment, a humanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. Ordinarily, the antibody will contain both the lightchain as well as at least the variable domain of a heavy chain. Theantibody also may include the CH1, hinge, CH2, CH3, and CH4 regions ofthe heavy chain. The humanized antibody can be selected from any classof immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and anyisotype, including IgG1, IgG2, IgG3 and IgG4. Usually the constantdomain is a complement fixing constant domain where it is desired thatthe humanized antibody exhibit cytotoxic activity, and the class istypically IgG.sub.1. Where such cytotoxic activity is not desirable, theconstant domain may be of the IgG.sub.2 class. The humanized antibodymay comprise sequences from more than one class or isotype, andselecting particular constant domains to optimize desired effectorfunctions is within the ordinary skill in the art. The framework and CDRregions of a humanized antibody need not correspond precisely to theparental sequences, e.g., the donor CDR or the consensus framework maybe mutagenized by substitution, insertion or deletion of at least oneresidue so that the CDR or framework residue at that site does notcorrespond to either the consensus or the import antibody. Suchmutations, however, will not be extensive. Usually, at least 75% of thehumanized antibody residues will correspond to those of the parentalframework region (FR) and CDR sequences, more often 90%, and mostpreferably greater than 95%. Humanized antibody can be produced usingvariety of techniques known in the art, including but not limited to,CDR-grafting (European Patent No. EP 239,400; International PublicationNo. WO 91/09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and5,585,089), veneering or resurfacing (European Patent Nos. EP 592,106and EP 519,596; Padlan, 1991, Molecular Immunology 28(4/5): 489-498;Studnicka et al., 1994, Protein Engineering 7(6): 805-814; and Roguskaet al., 1994, PNAS 91:969-973), chain shuffling (U.S. Pat. No.5,565,332), and techniques disclosed in, e.g., U.S. Pat. No. 6,407,213,U.S. Pat. No. 5,766,886, WO 9317105, Tan et al., J. Immunol. 169:1119-25(2002), Caldas et al., Protein Eng. 13(5): 353-60 (2000), Morea et al.,Methods 20(3): 267-79 (2000), Baca et al., J. Biol. Chem. 272(16):10678-84 (1997), Roguska et al., Protein Eng. 9(10): 895-904 (1996),Couto et al., Cancer Res. 55 (23 Supp): 5973s-5977s (1995), Couto etal., Cancer Res. 55(8): 1717-22 (1995), Sandhu J S, Gene 150(2): 409-10(1994), and Pedersen et al., J. Mol. Biol. 235(3): 959-73 (1994). Often,framework residues in the framework regions will be substituted with thecorresponding residue from the CDR donor antibody to alter, preferablyimprove, antigen binding. These framework substitutions are identifiedby methods well known in the art, e.g., by modeling of the interactionsof the CDR and framework residues to identify framework residuesimportant for antigen binding and sequence comparison to identifyunusual framework residues at particular positions. (See, e.g., Queen etal., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature332:323,)

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring that express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., migisepitope or immunogenic fragments thereof. Monoclonal antibodies directedagainst the antigen can be obtained from the immunized, transgenic miceusing conventional hybridoma technology. The human immunoglobulintransgenes harbored by the transgenic mice rearrange during B celldifferentiation, and subsequently undergo class switching and somaticmutation. Thus, using such a technique, it is possible to producetherapeutically useful IgG, IgA, IgM and IgE antibodies. For an overviewof this technology for producing human antibodies, see Lonberg andHuszar (1995, Int. Rev. Immunol. 13:65-93). For a detailed discussion ofthis technology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g.,International Publication Nos. WO 98/24893, WO 96/34096, and WO96/33735; and U.S. Pat. Nos. 5,413,923, 5,625,126, 5,633,425, 5,569,825,5,661,016, 5,545,806, 5,814,318, and 5,939,598. In addition, companiessuch as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.)and Medarex (Princeton, N.J.) can be engaged to provide human antibodiesdirected against a selected antigen using technology similar to thatdescribed above.

Further, the antibodies of the invention can, in turn, be utilized togenerate anti-idiotype antibodies that “mimic” a migis epitope (e.g.,cεmx.migis) using techniques well known to those skilled in the art.(See, e.g., Greenspan & Bona, 1989, FASEB J. 7(5): 437-444; andNissinoff, 1991, J. Immunol. 147(8): 2429-2438). For example, antibodiesof the invention which bind to and competitively inhibit the binding ofa migis epitope (as determined by assays well known in the art anddisclosed infra) to its ligands can be used to generate anti-idiotypesthat “mimic” migis binding domains and, as a consequence, bind to andneutralize migis containing proteins and/or its ligands. Suchneutralizing anti-idiotypes or Fab fragments of such anti-idiotypes canbe used in therapeutic regimens to neutralize migis containing proteins.The invention provides methods employing the use of polynucleotidescomprising a nucleotide sequence encoding an antibody of the inventionor a fragment thereof.

In one embodiment, the nucleotide sequence encoding an antibody thatspecifically binds a migis epitope is obtained and used to generate theantibodies of the invention. The nucleotide sequence can be obtained,for example, from sequencing hybridoma clone DNA. If a clone containinga nucleic acid encoding a particular antibody or an epitope-bindingfragment thereof is not available, but the sequence of the antibodymolecule or epitope-binding fragment thereof is known, a nucleic acidencoding the immunoglobulin may be chemically synthesized or obtainedfrom a suitable source (e.g., an antibody cDNA library, or a cDNAlibrary generated from, or nucleic acid, preferably poly A+ RNA,isolated from any tissue or cells expressing the antibody, such ashybridoma cells selected to express an antibody) by PCR amplificationusing synthetic primers that hybridize to the 3′ and 5′ ends of thesequence or by cloning using an oligonucleotide probe specific for theparticular gene sequence to identify, e.g., a cDNA clone from a cDNAlibrary that encodes the antibody. Amplified nucleic acids generated byPCR may then be cloned into replicable cloning vectors using any methodwell known in the art.

Once the nucleotide sequence of the antibody is determined, thenucleotide sequence of the antibody may be manipulated using methodswell known in the art for the manipulation of nucleotide sequences,e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.(see, Or example, the techniques described in Current Protocols inMolecular Biology, F. M. Ausubel et al., ed., John Wiley & Sons(Chichester, England, 1998); Molecular Cloning: A Laboratory Manual, 3ndEdition, J. Sambrook et al., ed., Cold Spring Harbor Laboratory Press(Cold Spring Harbor, N.Y., 2001); Antibodies: A Laboratory Manual, E.Harlow and D. Lane, ed., Cold Spring Harbor Laboratory Press (ColdSpring Harbor, N.Y., 1988); and Using Antibodies: A Laboratory Manual,E. Harlow and D. Lane, ed., Cold Spring Harbor Laboratory (Cold SpringHarbor, N.Y., 1999)), to generate antibodies having a different aminoacid sequence by, for example, introducing deletions, and/or insertionsinto desired regions of the antibodies.

In one embodiment, one or more substitutions are made within the Fcregion (e.g. supra) of an antibody able to specifically bind a migisepitope. In another embodiment, the amino acid substitutions modifybinding to one or more Fc ligand (e.g., FcγRs, C1q) and alter ADCCand/or CDC activity.

In a specific embodiment, one or more of the CDRs is inserted withinframework regions using routine recombinant DNA techniques. Theframework regions may be naturally occurring or consensus frameworkregions, specifically contemplated are human framework regions (see,e.g., Chothia et al., 1998, J. Mol. Biol. 278: 457-479 for a listing ofhuman framework regions). In one embodiment, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds to a migis epitope (e.g.,cεmx.migis). In another embodiment, as discussed supra, one or moreamino acid substitutions may be made within the framework regions, it iscontemplated that the amino acid substitutions improve binding of theantibody to its antigen. Additionally, such methods may be used to makeamino acid substitutions or deletions of one or more variable regioncysteine residues participating in an intrachain disulfide bond togenerate antibody molecules lacking one or more intrachain disulfidebonds. Other alterations to the polynucleotide are encompassed by thepresent invention and within the skill of the art.

5.4 Recombinant Expression of Antibodies

Recombinant expression of an antibody of the invention, derivative,analog or fragment thereof, (e.g., a heavy or light chain of an antibodyof the invention or a portion thereof or a single chain antibody of theinvention), requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody of theinvention has been obtained, the vector for the production of theantibody or fusion protein molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody or fusion protein encoding nucleotide sequence are describedherein. Methods that are well known to those skilled in the art can beused to construct expression vectors containing antibody codingsequences and appropriate transcriptional and translational controlsignals. These methods include, for example, in vitro recombinant DNAtechniques, synthetic techniques, and in vivo genetic recombination. Theinvention, thus, provides replicable vectors comprising a nucleotidesequence encoding an antibody of the invention, operably linked to apromoter. Such vectors may include the nucleotide sequence encoding theconstant region of the antibody molecule (see, e.g., InternationalPublication No. WO 86/05807; International Publication No. WO 89/01036;and U.S. Pat. No. 5,122,464) and the variable domain of the antibody ofthe invention may be cloned into such a vector for expression of thefall length antibody chain (e.g. heavy or light chain).

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention or fragments thereof, or a heavy or light chain thereof,or portion thereof, or a single chain antibody of the invention,operably linked to a heterologous promoter. In other embodiments for theexpression of double-chained antibodies, vectors encoding both the heavyand light chains may be co-expressed in the host cell for expression ofthe entire immunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibodies of the invention (see, e.g., U.S. Pat. No. 5,807,715).Such host-expression systems represent vehicles by which the codingsequences of interest may be produced and subsequently purified, butalso represent cells which may, when transformed or transfected with theappropriate nucleotide coding sequences, express an antibody of theinvention in situ. These include but are not limited to microorganismssuch as bacteria (e.g., E. coli and B. subtilis) transformed withrecombinant bacteriophage DNA, plasmid DNA or cosmid DNA expressionvectors containing antibody coding sequences; yeast (e.g., SaccharomycesPichia) transformed with recombinant yeast expression vectors containingantibody coding sequences; insect cell systems infected with recombinantvirus expression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHiK, 293, NS0, and 3T3 cells) harboringrecombinant expression constructs containing promoters derived from thegenome of mammalian cells (e.g., metallothionein promoter) or frommammalian viruses (e.g., the adenovirus late promoter; the vacciniavirus 7.5K promoter). Preferably, bacterial cells such as Escherichiacoli, and more preferably, eukaryotic cells, especially for theexpression of whole recombinant antibody, are used for the expression ofa recombinant antibody. For example, mammalian cells such as Chinesehamster ovary cells (CHO), in conjunction with a vector such as themajor intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., 1986, Gene 45:101; and Cockett et al., 1990,Bio/Technology 8:2). In a specific embodiment, the expression ofnucleotide sequences encoding antibodies that bind to the cεmx.migisepitope is regulated by a constitutive promoter, inducible promoter ortissue specific promoter.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodybeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of pharmaceutical compositions of anantibody, vectors that direct the expression of high levels of fusionprotein products that are readily purified may be desirable. Suchvectors include, but are not limited to, the E. coli expression vectorpUR278 (Ruther et al., 1983, EMBO 12:1791), in which the antibody orfusion protein coding sequence may be ligated individually into thevector in frame with the lac Z coding region so that a lac Z-fusionprotein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic AcidsRes. 13:3101-3109; Van Heeke & Schuster, 1989, J. Biol. Chem.24:5503-5509); and the like. pGEX vectors may also be used to expressforeign polypeptides as fusion proteins with glutathione 5-transferase(GST). In general, such fusion proteins are soluble and can easily bepurified from lysed cells by adsorption and binding to matrixglutathione agarose beads followed by elution in the presence of freeglutathione. The pGEX vectors are designed to include thrombin or factorXa protease cleavage sites so that the cloned target gene product can bereleased from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody in infected hosts (e.g., see Logan & Shenk,1984, Proc. Natl. Acad. Sci. USA 8 1:355-359). Specific initiationsignals may also be required for efficient translation of insertedantibody coding sequences. These signals include the ATG initiationcodon and adjacent sequences. Furthermore, the initiation codon must bein phase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (see, e.g., Bittner et al., 1987,Methods in Enzymol. 153:516-544).

The expression of an antibody may be controlled by any promoter orenhancer element known in the art. Promoters which may be used tocontrol the expression of the gene encoding an antibody or fusionprotein include, but are not limited to, the SV40 early promoter region(Bernoist and Chambon, 1981, Nature 290:304-310), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto, et al.,1980, Cell 22:787-797), the herpes thymidine kinase promoter (Wagner etal., 1981, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445), the regulatorysequences of the metallothionein gene (Brinster et al., 1982, Nature296:39-42), the tetracycline (Tet) promoter (Gossen et al., 1995, Proc.Nat. Acad. Sci. USA 89:5547-5551); prokaryotic expression vectors suchas the β-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl.Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer et al.,1983, Proc. Natl. Acad. Sci. U.S.A. 80:21-25; see also “Useful proteinsfrom recombinant bacteria” in Scientific American, 1980, 242:74-94);plant expression vectors comprising the nopaline synthetase promoterregion (HeiTera-Estrella et al., Nature 303:209-213) or the cauliflowermosaic virus 35S RNA promoter (Gardner et al., 1981, Nucl. Acids Res.9:2871), and the promoter of the photosynthetic enzyme ribulosebiphosphate carboxylase (Herrera-Estrella et al., 1984, Nature310:115-120); promoter elements from yeast or other fungi such as theGal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK(phosphoglycerol kinase) promoter, alkaline phosphatase promoter, andthe following animal transcriptional control regions, which exhibittissue specificity and have been utilized in transgenic animals:elastase I gene control region which is active in pancreatic acinarcells (Swift et al., 1984, Cell 38:639-646; Ornitz et al., 1986, ColdSpring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology7:425-515); insulin gene control region which is active in pancreaticbeta cells (Hanahan, 1985, Nature 315:115-122), immunoglobulin genecontrol region which is active in lymphoid cells (Grosschedl et al.,1984, Cell 38:647-658; Adames et al., 1985, Nature 318:533-538;Alexander et al., 1987, Mol. Cell. Biol. 7:1436-1444), mouse mammarytumor virus control region which is active in testicular, breast,lymphoid and mast cells (Leder et al., 1986, Cell 45:485-495), albumingene control region which is active in liver (Pinkert et al., 1987,Genes and Devel. 1:268-276), alpha-fetoprotein gene control region whichis active in liver (Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648;Hammer et al., 1987, Science 235:53-58; alpha I-antitrypsin gene controlregion which is active in the liver (Kelsey et al., 1987, Genes andDevel. 1:161-171), beta-globin gene control region which is active inmyeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias et al.,1986, Cell 46:89-94; myelin basic protein gene control region which isactive in oligodendrocyte cells in the brain (Readhead et al., 1987,Cell 48:703-712); myosin light chain-2 gene control region which isactive in skeletal muscle (Sani, 1985, Nature 314:283-286);neuronal-specific enolase (NSE) which is active in neuronal cells(Morelli et al, 1999, Gen. Virol. 80:571-83); brain-derived neurotrophicfactor (BDNF) gene control region which is active in neuronal cells(Tabuchi et al., 1998, Biochem. Biophysic. Res. Com. 253:818-823); glialfibrillary acidic protein (GFAP) promoter which is active in astrocytes(Gomes et al., 1999, Braz J Med Biol Res 32(5): 619-631; Morelli et al.,1999, Gen. Virol. 80:571-83) and gonadotropic releasing hormone genecontrol region which is active in the hypothalamus (Mason et al., 1986,Science 234:1372-1378).

Expression vectors containing inserts of a gene encoding an antibody canbe identified by three general approaches: (a) nucleic acidhybridization, (b) presence or absence of “marker” gene functions, and(c) expression of inserted sequences. In the first approach, thepresence of a gene encoding a peptide, polypeptide, protein or a fusionprotein in an expression vector can be detected by nucleic acidhybridization using probes comprising sequences that are homologous toan inserted gene encoding the peptide, polypeptide or protein,respectively. In the second approach, the recombinant vector/host systemcan be identified and selected based upon the presence or absence ofcertain “marker” gene functions (e.g., thymidine kinase activity,resistance to antibiotics, transformation phenotype, occlusion bodyformation in baculovirus, etc.) caused by the insertion of a nucleotidesequence encoding an antibody in the vector. For example, if thenucleotide sequence encoding the antibody is inserted within the markergene sequence of the vector, recombinants containing the gene encodingthe antibody insert can be identified by the absence of the marker genefunction. In the third approach, recombinant expression vectors can beidentified by assaying the gene product (e.g., antibody) expressed bythe recombinant. Such assays can be based, for example, on the physicalor functional properties of the antibody in in vitro assay systems,e.g., binding with anti-bioactive molecule.

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Expression from certainpromoters can be elevated in the presence of certain inducers; thus,expression of the genetically engineered fusion protein may becontrolled. Furthermore, different host cells have characteristic andspecific mechanisms for the translational and post-translationalprocessing and modification (e.g., glycosylation, phosphorylation ofproteins). Appropriate cell lines or host systems can be chosen toensure the desired modification and processing of the foreign proteinexpressed. For example, expression in a bacterial system will produce anunglycosylated product and expression in yeast will produce aglycosylated product. Eukaryotic host cells that possess the cellularmachinery for proper processing of the primary transcript (e.g.,glycosylation, and phosphorylation) of the gene product may be used.Such mammalian host cells include, but are not limited to, CHO, VERY,BHK, Hela, COS, MDCK, 293, 3T3, WI38, NSO, and in particular, neuronalcell lines such as, for example, SK-N-AS, SK-N-FI, SK-N-DZ humanneuroblastomas (Sugimoto et al., 1984, J. Natl. Cancer Inst. 73: 51-57),SK-N-SH human neuroblastoma (Biochim. Biophys. Acta, 1982, 704:450-460), Daoy human cerebellar medulloblastoma (He et al., 1992, CancerRes. 52: 1144-1148) DBTRG-05MG glioblastoma cells (Kruse et al., 1992,In Vitro Cell. Dev. Biol. 28A: 609-614), IMR-32 human neuroblastoma(Cancer Res., 1970, 30: 2110-2118), 1321NI human astrocytoma (Proc.Natl. Acad. Sci. USA, 1977, 74: 4816), MOG-G-CCM human astrocytoma (Br.J. Cancer, 1984, 49: 269), U87MG human glioblastoma-astrocytoma (ActaPathol. Microbiol. Scand., 1968, 74: 465-486), A172 human glioblastoma(Olopade et al., 1992, Cancer Res. 52: 2523-2529), C6 rat glioma cells(Benda et al., 1968, Science 161: 370-371), Neuro-2a mouse neuroblastoma(Proc. Natl. Acad. Sci. USA, 1970, 65: 129-136), NB41A3 mouseneuroblastoma (Proc. Natl. Acad. Sci. USA, 1962, 48: 1184-1190), SCPsheep choroid plexus (Bolin et al., 1994, J. Virol. Methods 48:211-221), G355-5, PG-4 Cat normal astrocyte (Haapala et al., 1985, J.Virol. 53: 827-833), Mpf ferret brain (Trowbridge et al., 1982, In Vitro18: 952-960), and normal cell lines such as, for example, CTX TNA2 ratnormal cortex brain (Radany et al., 1992, Proc. Natl. Acad. Sci. USA 89:6467-6471) such as, for example, CRL7030 and Hs578Bst. Furthermore,different vector/host expression systems may effect processing reactionsto different extents.

For long-term, high-yield production of recombinant proteins, stableexpression is often preferred. For example, cell lines which stablyexpress an antibody may be engineered. Rather than using expressionvectors that contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched medium, and then areswitched to a selective medium. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci thatin turn can be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines that express an antibodyof the invention that specifically binds to the cεmx.migis epitope. Suchengineered cell lines may be particularly useful in screening andevaluation of compounds that affect the activity of an that specificallybinds to cεmx.migis epitope.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can beemployed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance can be used as the basis of selection fordhfr, which confers resistance to methotrexate (Wigler et al., 1980,Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad.Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, whichconfers resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,1981, J. Mol. Biol. 150:1); and hygro, which confers resistance tohygromycin (SantetTe et al., 1984, Gene 30:147) genes.

Once a peptide, polypeptide, protein, antibody of the invention has beenproduced by recombinant expression, it may be purified by any methodknown in the art for purification of an antibody, for example, bychromatography (e.g., ion exchange, affinity, particularly by affinityfor the specific antigen after Protein A, and sizing columnchromatography), centrifugation, differential solubility, or by anyother standard technique for the purification of antibodies.

The expression levels of an antibody can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York,1987)). When a marker in the vector system expressing an antibody orfusion protein is amplifiable, increase in the level of inhibitorpresent in culture of host cell will increase the number of copies ofthe marker gene. Since the amplified region is associated with theantibody gene, production of the antibody will also increase (Crouse etal., 1983, Mol. Cell. Biol. 3:257).

The host cell may be co-transfected with two expression vectors of theinvention. For example, the first vector encoding a heavy chain derivedpolypeptide and the second vector encoding a light chain derivedpolypeptide. The two vectors may contain identical selectable markers,which enable equal expression of heavy and light chain polypeptides.Alternatively, a single vector may be used which encodes, and is capableof expressing, a fusion protein or both heavy and light chainpolypeptides. The coding sequences for the fusion protein or heavy andlight chains may comprise cDNA or genomic DNA.

5.5 Biological Assays

The binding specificity, affinity and functional activity of an antibodyof the invention can be characterized in various in vitro binding andcell adhesion assays known in the art, including but limited to, ELISAWestern Blot analysis, cell surface staining, inhibition ofligand-receptor interactions, flow cytometric analysis and thosedisclosed in International Publication Nos. WO 04/014292, WO 03/094859,WO 04/069264, WO 04/028551, WO 03/004057, WO 03/040304, WO 00/78815, WO02/070007 and WO 03/075957, U.S. Pat. Nos. 5,795,734, 6,248,326 and6,472,403, Pecheur et al., 2002, FASEB J. 16(10): 1266-1268; Almed etal., The Journal of Histochemistry & Cytochemistry 50:1371-1379 (2002).For example, the binding affinity, specificity and the off-rate ofantibody of the invention can be determined by a competitive bindingassay, by measuring the inhibitory activity of antibody of the inventionon binding to a migis epitope. One example of a competitive bindingassay is a radioimmunoassay comprising the incubation of labeled peptidecomprising a migis epitope (e.g., 3H or 125I) with the antibody of theinvention in the presence of increasing amounts of unlabeled peptide,and the detection of the antibody bound to the labeled peptide. Theaffinity of an Fc variant for a migis epitope and the binding off-ratescan be determined from the data by scatchard plot analysis. Competitionwith a second antibody can also be determined using radioimmunoassays.In this case, a peptide comprising a migis epitope is incubated with anantibody of the invention conjugated to a labeled compound (e.g., 3H or125I) in the presence of increasing amounts of a second unlabeledmonoclonal antibody.

The kinetic parameters of an antibody of the invention may also bedetermined using any surface plasmon resonance (SPR) based assays knownin the art. For a review of SPR-based technology see Mullet et al.,2000, Methods 22: 77-91; Dong et al., 2002, Review in Mol. Biotech., 82:303-23; Fivash et al., 1998, Current Opinion in Biotechnology 9: 97-101;Rich et al., 2000, Current Opinion in Biotechnology 11: 54-61.Additionally, any of the SPR instruments and SPR based methods formeasuring protein-protein interactions described in U.S. Pat. Nos.6,373,577; 6,289,286; 5,322,798; 5,341,215; 6,268,125 are contemplatedin the methods of the invention.

The binding specificity of antibody of the invention to a migis peptidecan be assessed by any method known in the art including but not limitedto, measuring binding to a migis epitope and its crossreactivity toother migis-containing peptides.

The ability of an antibody of the invention to bind to a migis epitopepresent on a mIg can be determined by methods well known in the art suchas flow cytometric analysis and other cell staining techniques includingbut not limited to immunohistochemistry.

It is contemplated that the protocols and formulations of the inventionare tested in vitro, and then in vivo, for the desired therapeutic orprophylactic activity, prior to use in humans. For example, assays whichcan be used to determine whether administration of a specifictherapeutic protocol, formulation or combination therapy of theinvention is indicated, include in vitro cell culture assays in which atarget cell is grown in culture, and exposed to or otherwise contactedwith a formulation of the invention, and the effect of such aformulation upon the tissue sample is observed.

In particular, the ability of any particular antibody to mediate lysisof the target cell by ADCC or CDC can be assayed. To assess ADCCactivity an antibody of interest is added to target cells in combinationwith immune effector cells, which may be activated by the antigenantibody complexes resulting in cytolysis of the target cell. Cytolysisis generally detected by the release of label (e.g. radioactivesubstrates, fluorescent dyes or natural intracellular proteins) from thelysed cells. Useful effector cells for such assays include peripheralblood mononuclear cells (PBMC) and Natural Killer (NK) cells. Specificexamples of in vitro ADCC assays are described in Wisecarver et al.,1985 79:277-282; Bruggemann et al., 1987, J Exp Med 166:1351-1361;Wilkinson et al., 2001, J Immunol Methods 258:183-191; Patel et al.,1995 J Immunol Methods 184:29-38 and herein (see Example 3).Alternatively, or additionally, ADCC activity of the antibody ofinterest may be assessed in vivo, e.g., in a animal model such as thatdisclosed in Clynes et al., 1998, PNAS USA 95:652-656. To assess the CDCactivity of an antibody, a CDC assay, e.g. as described inGazzano-Santoro et al., 1996, J Immunol. Methods, 202:163, may beperformed.

Prophylactic or therapeutic agents can be tested in suitable animalmodel systems prior to testing in humans, including but not limited toin rats, mice, chicken, cows, monkeys, rabbits, hamsters, etc. Forexample one of the most relevant animal systems for the study of asthmais the Rhesus Monkey Model. The rhesus monkey model make use of the factthat a small number of rhesus monkeys, which have been infected with thenematode Ascaris suum, develop sensitivity to extract of ascaris. Whenthese sensitive monkeys are given spray containing ascaris antigen, theydevelop breathing problems resembling asthma. Patterson, R., J. Clini.Invest. 57: 586-593 (1976). The antibodies of this invention can betested in the asthma/rhesus monkey model system. The ascaris sensitivemonkeys are given the experimental treatment or control treatment andmeasurements are made to determine the clinical outcome of treatment.Measurements include quantification of one or more of the followingindicators, asthma symptoms upon ascaris challenge, the level ofcirculating IgE, the of circulating IgE-bearing B cells and the IgEdensity on basophils.

5.6 Prophylactic and Therapeutic Uses

As discussed above, agents that immunospecifically bind a migis epitopecan be utilized for the prevention, management, treatment oramelioration of B-cell mediated diseases and disorders including, thoseresulting from or associated with monoclonal expansion of B-cells, andin particular those mediated by IgE.

Diseases and disorders mediated by IgE include those associated with thebinding of IgE to FcεRI such as, for example, allergic disease caused byIgE antibodies and mast cell mediators including but not limited toatopic diseases such as allergic rhinitis, allergic asthma, includingasthma associated with specific antigenic factors such as seasonalpollens (grass: rye, timothy, ragweed) and tree (birch), perennialallergens such as dust mite, animal danders, feathers and fungal sporesand occupational antigens such as detergents and metals as well asasthma associated with non-antigen specific factors such as infection,irritants such as smoke, fumes, diesel exhaust particles and sulphurdioxide, asthma associated with airway cooling (exercise, cold airtemperatures) and emotional stress; atopic dermatitis and allergicgastroenteropathy as well as anaphylactic diseases including systemicanaphylaxis and reactions to proteins in foods (e.g., peanut), venom,vaccines, hormones, antiserum, enzymes and latex, reactions to haptensincluding antibiotics, muscle relaxants, vitamins, cytotoxins andopiates and reactions to polysaccharides such as dextran, iron dextranand polygeline and other anaphylactic diseases or disorders such asurticaria-angioedema.

In addition, certain gastro-intestinal inflammatory disorders are knownto be IgE-mediated. Such IgE-mediated gastro-intestinal inflammatorydisorders can be broadly defined as intractable chronic response to a toa variety of insults, such as those caused by injury or infection whichare characterized by, or results from pathology affected by IgE.Particular disorders included within the scope of IgE-mediatedgastro-intestinal inflammatory disorders includes inflammatory boweldisease (e.g., Crohn's disease, ulcerative colitis, indeterminatecolitis and infectious colitis), gastroenteropathy, enteritis, mucositis(e.g., oral mucositis, gastrointestinal mucositis, nasal mucositis andproctitis), necrotizing enterocolitis and esophagitis).

Diseases and disorders or associated with B-cell expansion diseasesinclude, for example, hyper IgE syndrome (Job's disease), posttransplant lymphoproliferative disorder (PTLD), monocolonal gamiiopathof unknown significance (MGUS), Waldenstrom Macroglubulinemia,neuropathy, nephropathy, myelomas, inflammatory and autoimmune diseasessuch as Rheumatoid arthritis and Lupus.

Diseases and disorders which can be prevented, treated or inhibited byadministering an effective amount of one or more antibodies of theinvention include, but are not limited to, asthma, autoimmune disorders(e.g., lupus, rheumatoid arthritis, multiple sclerosis, myastheniagravis, Hashimoto's disease, and immunodeficiency syndrome),inflammatory disorders (e.g., asthma, allergic disorders, and rheumatoidarthritis), infectious diseases (e.g., AIDS), and proliferativedisorders (e.g., leukemia, carcinoma, and lymphoma). In a specificembodiment, the subject antibodies will be used to treat asthma. Inanother embodiment, the subject antibodies will be used to treatdiseases involving mucin production as a major component of pathology.Such diseases include cystic fibrosis, emphysema and COPD by way ofexample.

5.7 Formulations and Administration

As described above, the present invention relates to the use of agentsthat specifically bind a migis epitope for the prevention, management,treatment or amelioration of a B-cell mediated disease or disorder.Accordingly, the present invention provides formulations (e.g., apharmaceutical composition) comprising one or more antibodies of theinvention that specifically bind to a migis epitope (also referred toherein as “formulation(s) of the invention” or simply “formulation(s)”).In specific embodiments, the agent specifically binds a cεmx.migisepitope and inhibits IgE production. Accordingly, it is contemplatedthat formulation comprising an agent that specifically binds to acsmx.migis epitope is useful for the prevention, management, treatmentor amelioration of an IgE-mediated disease (e.g., allergies) or one ormore symptoms thereof.

In one embodiment, formulations (e.g., a pharmaceutical composition)comprising one or more antibodies of the invention are liquidformulations (referred to herein as “liquid formulation(s)” which arespecifically encompassed by the more generic terms “formulation(s) ofthe invention” and “formulation(s)”). In a specific embodiment, theliquid formulations are substantially free of surfactant and/orinorganic salts. In another specific embodiment, the liquid formulationshave a pH ranging from about 5.0 to about 7.0, about 5.5 to about 6.5,or about 5.8 to about 6.2, or about 6.0. In another specific embodiment,the liquid formulations have a pH ranging from 5.0 to 7.0, 5.5 to 6.5,or 5.8 to 6.2, or 6.0. In yet another specific embodiment, the liquidformulations comprise histidine at a concentration ranging from about 1mM to about 100 mM, or from about 5 mM to about 50 mM, or about 10 mM toabout 25 mM. In still another specific embodiment, the liquidformulations comprise histidine at a concentration ranging from 1 mM to100 mM, or from 5 mM to 50 mM, or 10 mM to 25 mM

In another embodiment, the liquid formulations have a concentration ofone or more antibodies of the invention that is about 50 mg/ml, about 75mg/ml, about 100 mg/ml, about 125 mg/ml, about 150 mg/ml, about 175mg/ml, about 200 mg/ml, about 225 mg/ml, about 250 mg/ml, about 275mg/ml, or about 300 mg/ml. In another embodiment, the liquidformulations have a concentration of one or more antibodies of theinvention that is 50 mg/ml, 75 mg/ml, 100 mg/ml, 125 mg/ml, 150 mg/ml,175 mg/ml, 200 mg/ml, 225 mg/ml, 250 mg/ml, 275 mg/ml, or 300 mg/ml. Instill another embodiment, the liquid formulations should exhibit one, ormore of the following characteristics, stability, low to undetectablelevels of antibody fragmentation and/or aggregation, very little to noloss of the biological activities of the antibodies or antibodyfragments during manufacture, preparation, transportation, and storage.In certain embodiments the liquid formulations lose less than 50%, orless than 30%, or less than 20%, or less than 10% or even less than 5%or 1% of the antibody activity within 1 year storage under suitableconditions at about 4° C. The activity of an antibody can be determinedby a suitable antigen-binding or effector function assay for therespective antibody. In yet another embodiment, the liquid formulationsare of low viscosity and turbidity. In a particular embodiment, theliquid formulations have a viscosity of less than 10.00 cP at anytemperature in the range of 1 to 26° C. Viscosity can be determined bynumerous method well known in the art. For example, the viscosity of apolypeptide solution can be measured using a ViscoLab 4000 ViscometerSystem (Cambridge Applied Systems) equipped with a ViscoLab Piston(SN:7497, 0.3055″, 1-20 cP) and S6S Reference Standard (KoehlerInstrument Company, Inc.) and connected to a water bath to regulate thetemperature of the samples being analyzed. The sample is loaded into thechamber at a desired starting temperature (e.g., 2° C.) and the pistonlowered into the sample. After sample was equilibrated to thetemperature of the chamber, measurement is initiated. The temperature isincreased at a desired rate to the desired final temperature (e.g., >25°C.). And the viscosity over time is recorded.

It is contemplated that the liquid formulations may further comprise oneor more excipients such as a saccharide, an amino acid (e.g. arginine,lysine, and methionine) and a polyol. Additional descriptions andmethods of preparing and analyzed liquid formulations can be found, forexample, in PCT publications WO 03/106644; WO 04/066957; WO 04/091658.

In one embodiment the formulations (e.g., liquid formulations) of theinvention are pyrogen-free formulations which are substantially free ofendotoxins and/or related pyrogenic substances. Endotoxins includetoxins that are confined inside a microorganism and are released whenthe microorganisms are broken down or die. Pyrogenic substances alsoinclude fever-inducing, thermostable substances (glycoproteins) from theouter membrane of bacteria and other microorganisms. Both of thesesubstances can cause fever, hypotension and shock if administered tohumans. Due to the potential harmful effects, it is advantageous toremove even low amounts of endotoxins from intravenously administeredpharmaceutical drug solutions. The Food & Drug Administration (“FDA”)has set an upper limit of 5 endotoxin units (EU) per dose per kilogrambody weight in a single one hour period for intravenous drugapplications (The United States Pharmacopeial Convention, PharmacopeialForum 26 (1):223 (2000)). When therapeutic proteins are administered inamounts of several hundred or thousand milligrams per kilogram bodyweight, as can be the case with monoclonal antibodies, it isadvantageous to remove even trace amounts of endotoxin. In oneembodiment, endotoxin and pyrogen levels in the composition are lessthen 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.

It will be apparent to one skilled in the art that a formulationcomprising one or more antibodies of the invention to be administered toa subject (e.g., a human) in need thereof should be formulated in apharmaceutically-acceptable excipient. Examples of formulations,pharmaceutical compositions in particular, of the invention include butare not limited to those disclosed in PCT publications WO 02/070007, WO03/075957 and WO 04/066957. Briefly, the excipient that is included withthe antibodies of the invention in these formulations (e.g., liquidformulations) can be selected based on the expected route ofadministration of the formulations in therapeutic applications. Theroute of administration of the formulations depends on the condition tobe treated. For example, intravenous injection may be preferred fortreatment of a systemic disorder such as a lymphatic cancer or a tumorwhich has metastasized. The dosage of the formulations to beadministered can be determined by the skilled artisan without undueexperimentation in conjunction with standard dose-response studies.Relevant circumstances to be considered in making those determinationsinclude the condition or conditions to be treated, the choice offormulations to be administered, the age, weight, and response of theindividual patient, and the severity of the patient's symptoms. Forexample, the actual patient body weight may be used to calculate thedose of the antibodies of the invention in these formulations inmilliliters (mL) to be administered. There may be no downward adjustmentto “ideal” weight. In such a situation, an appropriate dose may becalculated by the following formula:

Dose (mL)=[patient weight (kg)×dose level (mg/kg)/drug concentration(mg/mL)]

Depending on the condition, the formulations can be administered orally,parenterally, intramuscularly, intranasally, vaginally, rectally,lingually, sublingually, buccally, intrabuccally, intravenously,cutaneously, subcutaneously and/or transdermally to the patient.

Accordingly, formulations designed for oral, parenteral, intramuscular,intranasal, vaginal, rectal, lingual, sublingual, buccal, intrabuccal,intravenous, cutaneous, subcutaneous and/or transdermal administrationcan be made without undue experimentation by means well known in theart, for example, with an inert diluent or with an edible carrier. Theformulations may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, theformulations of the present invention may be incorporated withexcipients and used in the form of tablets, troches, capsules, elixirs,suspensions, syrups, wafers, chewing gums, and the like.

Tablets, pills, capsules, troches and the like may also contain binders,recipients, disintegrating agent, lubricants, sweetening agents, and/orflavoring agents. Some examples of binders include microcrystallinecellulose, gum tragacanth and gelatin. Examples of excipients includestarch and lactose. Some examples of disintegrating agents includealginic acid, cornstarch, and the like. Examples of lubricants includemagnesium stearate and potassium stearate. An example of a glidant iscolloidal silicon dioxide. Some examples of sweetening agents includesucrose, saccharin, and the like. Examples of flavoring agents includepeppermint, methyl salicylate, orange flavoring, and the like. Materialsused in preparing these various formulations should be pharmaceuticallypure and non-toxic in the amounts used.

The formulations of the present invention can be administeredparenterally, such as, for example, by intravenous, intramuscular,intrathecal and/or subcutaneous injection. Parenteral administration canbe accomplished by incorporating the formulations of the presentinvention into a solution or suspension. Such solutions or suspensionsmay also include sterile diluents, such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycoland/or other synthetic solvents. Parenteral formulations may alsoinclude antibacterial agents, such as, for example, benzyl alcoholand/or methyl parabens, antioxidants, such as, for example, ascorbicacid and/or sodium bisulfite, and chelating agents, such as EDTA.Buffers, such as acetates, citrates and phosphates, and agents for theadjustment of tonicity, such as sodium chloride and dextrose, may alsobe added. The parenteral preparation can be enclosed in ampules,disposable syringes and/or multiple dose vials made of glass or plastic.Rectal administration includes administering the formulation into therectum and/or large intestine. This can be accomplished usingsuppositories and/or enemas. Suppository formulations can be made bymethods known in the art. Transdermal administration includespercutaneous absorption of the formulation through the skin. Transdermalformulations include patches, ointments, creams, gels, salves, and thelike. The formulations of the present invention can be administerednasally to a patient. As used herein, nasally administering or nasaladministration includes administering the formulations to the mucousmembranes of the nasal passage and/or nasal cavity of the patient.

In certain embodiments, the formulations (e.g., liquid formulations) areadministered to the mammal by subcutaneous (i.e., beneath the skin)administration. For such purposes, the formulations may be injectedusing a syringe. However, other devices for administration of theformulations are available such as injection devices (e.g. theInject-ease_and Genject_devices), injector pens (such as the GenPen™);auto-injector devices, needleless devices (e.g., MediJector andBioJector); and subcutaneous patch delivery systems.

In another aspect of the invention there is provided a slow releaseformulations. In a specific embodiment, a slow release formulationcomprises a liquid formulation. Slow release formulations may beformulated from a number of agents including, but not limited to,polymeric nano or microparticles and gels (e.g., a hyaluronic acid gel).Besides convenience, slow release formulations offer other advantagesfor delivery of protein drugs including protecting the protein (e.g.,antibody of the invention) over an extended period from degradation orelimination, and the ability to deliver the protein locally to aparticular site or body compartment thereby lowering overall systemicexposure.

The present invention, for example, also contemplates injectable depotformulations in which the protein (e.g., antibody of the invention) isembedded in a biodegradable polymeric matrix. Polymers that may be usedinclude, but are not limited to, the homo- and co-polymers of lactic andglycolic acid (PLGA). PLGA degrades by hydrolysis to ultimately give theacid monomers and is chemically unreactive under the conditions used toprepare, for example, microspheres and thus does not modify the protein.After subcutaneous or intramuscular injection, the protein is releasedby a combination of diffusion and polymer degradation. By using polymersof different composition and molecular weight, the hydrolysis rate canbe varied thereby allowing release to last from days to months. In afurther aspect the present invention provides a nasal spray formulation.In a specific embodiment, a nasal spray formulation comprises the liquidformulation of the present invention.

The formulations of the invention may be used in accordance with themethods of the invention for the prevention, management, treatment oramelioration of B-cell mediated diseases including but not limited toallergic diseases, myelomas, diseases caused by monoclonal expansion ofB-cells, autoimmune and inflammatory diseases (in particular aIgE-mediated disease) or one or more symptoms thereof. In oneembodiment, the formulations of the invention are sterile and insuitable form for a particular method of administration to a subjectwith a B-cell mediated diseases including but not limited to allergicdiseases, myelomas, diseases caused by monoclonal expansion of B-cells,autoimmune and inflammatory diseases (in particular a IgE-mediateddisease).

The formulations of the invention may comprise other active ingredientsincluding, but are not limited to, one or more of inhaled asthmamedication, such as but not limited to an asthma related therapeutic, aTNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, ananalgesic, an anesthetic, a sedative, a local anethetic, a neuromuscularblocker, an antimicrobial, an antipsoriatic, a corticosteriod, ananabolic steroid, an asthmua related agent, a mineral, a nutritional, athyroid agent, a vitamin, a calcium related hormone, an antidiarrheal,an antitussive, an antiemetic, an antiulcer, a laxative, ananticoagulant, an erythropieitin, a filgrastim, a sargramostim, animmunization, an immunoglobulin, an immunosuppressive, a growth hormone,a hormone replacement drug, an estrogen receptor modulator, a mydriatic,a cycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, astimulant, donepezil, tacrine, an asthma medication, a beta agonist, aninhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn,an epinephrine or analog, domase alpha, a cytokine, or a cytokineantagonist.

In particular, asthma-related compositions of the invention canoptionally further comprise at least one selected from an asthma-relatedtherapeutic, a TNF antagonist (e.g., but not limited to a TNF Ig derivedprotein or fragment, a soluble TNF receptor or fragment, fusion proteinsthereof, or a small molecule TNF antagonist), an antirheumatic, a musclerelaxant, a narcotic, a non-steroid anti-inflammatory drug (NSAID), ananalgesic, an anesthetic, a sedative, a local anethetic, a neuromuscularblocker, an antimicrobial (e.g., aminoglycoside, an antifungal, anantiparasitic, an antiviral, a carbapenem, cephalosporin, afluororquinolone, a macrolide, a penicillin, a sulfonamide, atetracycline, another antimicrobial), an antipsoriatic, acorticosteriod, an anabolic steroid, an asthma related agent, a mineral,a nutritional, a thyroid agent, a vitamin, a calcium related hormone, anantidialrheal, an antitussive, an antiemetic, an antiulcer, a laxative,an anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgrastim(e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), animmunization, an immunoglobulin, an immunosuppressive (e.g.,basiliximab, cyclosporine, daclizumab), a growth hormone, a hormonereplacement drug, an estrogen receptor modulator, a mydriatic, acycloplegic, an alkylating agent, an antimetabolite, a mitoticinhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, anantipsychotic, an anxiolytic, a hypnotic, inhaled glucocorticosteroids,a sympathomimetic, a stimulant, donepezil, tacrine, an asthmamedication, a beta agonist, an inhaled steroid, a leukotriene inhibitor,a methylxanthine, a cromolyn, an epinephrine or analog, domase alpha(Pulmozyme), a cytokine or a cytokine antagonist. Suitable amounts anddosages are well known in the art. See, e.g., Wells et al., eds.,Pharmacotherapy Handbook, 2.sup.nd Edition, Appleton and Lange,Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000).

The invention provides methods for preventing, managing, treating orameliorating B-cell mediated diseases including but not limited toallergic diseases, myelomas, diseases caused by monoclonal expansion ofB-cells, autoimmune and inflammatory diseases (in particular aIgE-mediated disease) or one or more symptoms thereof, said methodcomprising: (a) administering to a subject in need thereof a dose of aprophylactically or therapeutically effective amount of a formulationcomprising one or more antibodies of the invention, that specificallybind to a migis epitope and (b) administering one or more subsequentdoses of said formulation, to maintain a plasma concentration of theantibody of the invention at a desirable level (e.g., about 0.1 to about100 μg/ml). In a specific embodiment, the plasma concentration of theantibody of the invention is maintained at 10 μg/ml, 15 μg/ml, 20 μg/ml,25 μg/ml, 30 μg/ml, 35 μg/ml, 40 μg/ml, 45 μg/1 ml or 50 μg/ml. In aspecific embodiment, said effective amount of the antibody of theinvention to be administered is between at least 1 mg/kg and 100 mg/kgper dose. In another specific embodiment, said effective amount of theantibody of the invention to be administered is between at least 1 mg/kgand 20 mg/kg per dose. In another specific embodiment, said effectiveamount of the antibody of the invention to be administered is between atleast 4 mg/kg and 10 mg/kg per dose. In yet another specific embodiment,said effective amount of the antibody of the invention to beadministered is between 50 mg and 250 mg per dose. In still anotherspecific embodiment, said effective amount of the antibody of theinvention to be administered is between 100 mg and 200 mg per dose.

The present invention provides kits comprising one or more antibodies ofthe invention that specifically bind to a migis epitope conjugated orfused to a detectable agent, therapeutic agent or drug, in one or morecontainers, for use in the prevention, treatment, management,amelioration, detection, monitoring or diagnosis of B-cell mediateddiseases including but not limited to allergic diseases, myelomas,diseases caused by monoclonal expansion of B-cells, autoimmune andinflammatory diseases, in particular a IgE-mediated disease or disorder.“IgE-mediated disorder” and “IgE mediated disease” means a condition ordisease which is characterized by the overproduction and/orhypersensitivity to the immunoglobulin IgE. Specifically, IgE-mediateddisorders include conditions associated with anaphylactichypersensitivity and atopic allergies, including for example: asthma,allergic rhinitis and conjunctivitis (hay fever), eczema, urticaria andfood allergies.

The invention also provides kits comprising one or more antibodies ofthe invention that specifically binds to a migis peptide in a first vialand one or more prophylactic or therapeutic agents, other than anantibody of the invention, in a second vial for use in the prevention,treatment, management, amelioration, detection, monitoring or diagnosisof B-cell mediated diseases including but not limited to allergicdiseases, myelomas, diseases caused by monoclonal expansion of B-cells,autoimmune and inflammatory diseases, in particular a IgE-mediateddisease. The invention also provides kits comprising one or moreantibody of the invention that specifically binds to a migis peptideconjugated or fused to a therapeutic agent or drug in a first vial andone or more prophylactic or therapeutic agents, other than an antibodyof the invention, in a second vial for use in the prevention, treatment,management, amelioration, detection, monitoring or diagnosis of B-cellmediated diseases including but not limited to allergic diseases,myelomas, diseases caused by monoclonal expansion of B-cells, autoimmuneand inflammatory diseases, in particular a IgE-mediated disease. Thekits may further comprise packaging materials and/or instructions.

TABLE 1 Legend for Sequence Listing SEQ ID NO: Description 1 Humanε-migis amino acid sequence 2 Human μ-migis amino acid sequence 3 Humanphosphoinositide binding protein epitope amino acid sequence 4 HumanKIAA1227 epitope amino acid sequence 5 Human cεmx.migis peptide sequence6 Portion of Human cεmx peptide sequence 7 D5 antibody V_(L) nucleotidesequence 8 D5 antibody V_(H) nucleotide sequence 9 D5 antibody V_(L)amino acid sequence 10 D5 antibody V_(H) amino acid sequence 11 D5 V_(L)CDR1 amino acid sequence 12 D5 V_(L) CDR2 amino acid sequence 13 D5V_(L) CDR3 amino acid sequence 14 D5 V_(H) CDR1 amino acid sequence 15D5 V_(H) CDR2 amino acid sequence 16 D5 V_(H) CDR3 amino acid sequence17 ADWPGPP ELDVCVEEAEGEAPW 18 DWPGPP ELDVCVEEAEGEAPW 19 WPGPPELDVCVEEAEGEAPW 20 PGPP ELDVCVEEAEGEAPW 21 GPP ELDVCVEEAEGEAPW 22 PPELDVCVEEAEGEAPW 23 P ELDVCVEEAEGEAPW 24 RADWPGPP ELDVCVEEAEGEAP 25RADWPGPP ELDVCVEEAEGEA 26 RADWPGPP ELDVCVEEAEGE 27 RADWPGPP ELDVCVEEAEG28 RADWPGPP ELDVCVEEAE 29 RADWPGPP ELDVCVEEA 30 RADWPGPP ELDVCVEE 31RADWPGPP ELDVCVE 32 RADWPGPP ELDVCV 33 RADWPGPP ELDVC 34 RADWPGPP ELDV35 RADWPGPP ELD 36 RADWPGPP EL 37 RADWPGPP E 38 ADWPGPP ELDVCVEEAEGEAP39 DWPGPP ELDVCVEEAEGEA 40 GPP ELD 41 PGPP ELDV 42 PGPP ELD 43 GPP ELDV44 WPGPP ELDVC 45 PGPP ELDVC 46 WPGPP ELDV 47 Human δ-migis amino acidsequence 48 Human γ-migis amino acid sequence 49 Human α-migis aminoacid sequence 50 A1c antibody V_(L) nucleotide sequence 51 A1c antibodyV_(H) nucleotide sequence 52 A1c antibody V_(L) amino acid sequence 53A1c antibody V_(H) amino acid sequence 54 A1c V_(L) CDR1 amino acidsequence 55 A1c V_(L) CDR2 amino acid sequence 56 A1c V_(L) CDR3 aminoacid sequence 57 A1c V_(H) CDR1 amino acid sequence 58 A1c V_(H) CDR2amino acid sequence 59 A1c V_(H) CDR3 amino acid sequence 60 B1 antibodyV_(L) nucleotide sequence 61 B1 antibody V_(H) nucleotide sequence 62 B1antibody V_(L) amino acid sequence 63 B1 antibody V_(H) amino acidsequence 64 B1 V_(L) CDR1 amino acid sequence 65 B1 V_(L) CDR2 aminoacid sequence 66 B1 V_(L) CDR3 amino acid sequence 67 B1 V_(H) CDR1amino acid sequence 68 B1 V_(H) CDR2 amino acid sequence 69 B1 V_(H)CDR3 amino acid sequence 70 F4 antibody V_(L) nucleotide sequence 71 F4antibody V_(H) nucleotide sequence 72 F4 antibody V_(L) amino acidsequence 73 F4 antibody V_(H) amino acid sequence 74 F4 V_(L) CDR1 aminoacid sequence 75 F4 V_(L) CDR2 amino acid sequence 76 F4 V_(L) CDR3amino acid sequence 77 F4 V_(H) CDR1 amino acid sequence 78 F4 V_(H)CDR2 amino acid sequence 79 F4 V_(H) CDR3 amino acid sequence 80 D9antibody V_(H) nucleotide sequence 81 D9 antibody V_(H) amino acidsequence 82 D9 V_(H) CDR1 amino acid sequence 83 D9 V_(H) CDR2 aminoacid sequence 84 D9 V_(H) CDR3 amino acid sequence 85 scrambled form ofε-migis 86 IgG migis peptide with 8 additional amino acids at N-terminus87 IgM migis peptide with 8 additional amino acids at N-tenninus ε-migisamino acid residues are underlined cεmx amino acid residues are bolded

6. EXAMPLES

The invention is now described with reference to the following examples.These examples are provided for the purpose of illustration only and theinvention should in no way be construed as being limited to theseexamples but rather should be construed to encompass any and allvariations which become evident as a result of the teachings providedherein.

6.1 Example 1 Development of Human Anti 2-migis Antibodies

15 unique phage clones that bound the ε-migis peptide (SEQ ID NO.:1)were isolated from a naïve human Fab phage display library usingstandard soluble and immobilized antigen panning techniques. All of theisolated antibodies specifically bound the ε-migis peptide but not to ascrambled peptide by ELISA. However, only one antibody, designated“A1c,” (see FIG. 13) bound to cells expressing membrane anchored IgE.Upon further investigation it was determined that A1c also bound toother cell types including membrane IgM, IgA expressing cells andT-cells (data not shown). Examination of the ε-migisamino acid sequencerevealed that part of the epitope is shared by other proteins includingphosphoinositide binding protein, recently found to be the receptor foranthrax PA (Lu Q, et al., Proc Natl Acad Sci USA. 2004, 101:17246-17251,and unknown hypothetical protein, KIAA1227 (see FIG. 1B). A peptidecorresponding to the region of the phosphoionositide binding proteinthat is similar to ε-migis (peptide vi in “Materials and Methods”) wassynthesized and used in an ELISA assay for examining binding of the antiε-migisantibody. As shown in FIG. 7A it was found that the anti ε-migisantibody A1c bound to this peptide nearly as efficiently as it did toε-migis peptide. In contrast antibodies that bound to cεmx-migis (seebelow) did not bind to this peptide. Together, these results suggestthat the ε-migis peptide may not be an effective target for mIgEspecific antibody binding.

To obtain human antibody clones with greater specificity a secondpeptide, designated “cεmx.migis” (SEQ ID NO:5) containing an additionaleight amino acids from the cεmx region (FIG. 2) of the long isoform ofhuman membrane anchored ε-chain, was used for isolation of additionalclones from the human antibody phage display library. After three roundsof panning, phage isolates from about 2500 individual bacterial colonieswere screened by peptide ELISA. The binding characteristics of ˜364isolated clones positive for binding to the cεmx.migis peptide wasexamined. ELISA studies demonstrated that roughly two thirds of theclones bound both the cεmx.migs and ε-migis peptides, the remainingthird (˜108) bound only the cεmx.migs peptide. These did not bind toε-migis or other related peptides that were tested (data not shown). 20were randomly chosen for further study and converted to full IgG. FACSanalysis of the cεmx.migis specific clones revealed that like theε-migis specific clones initially isolated the cεmx-migis antibodiesfell into two categories. About 25% did not bind to cells expressingmembrane anchored IgE while ˜75% were not specific for cells expressingmembrane anchored IgE (i.e., bound to all human cells testedirrespective of whether mIgE was expressed or not, similar to cloneA1c).

Using standard screening methods both the ε-migis and the cεmx.migispeptide specific antibodies either bound to human cells irrespective ofmIgE expression or did not bind to cells at all. Together, these studiesindicate that one predominant epitope of the ε-migis peptide is sharedby at least one widely expressed cell surface protein (designated“shared-epitope”) and that an ε-migis-dependent epitope of thecεmx.migis peptide is either hidden or absent when the ε-chain ispresent on the cell surface (designated “hidden-epitope”). Usingstandard panning and screening methods the majority of clones isolatedrecognized these predominant epitopes. For example, A1c binds theshared-epitope while CP1-B1 (also designated “B1”, see FIG. 14) bindsthe hidden-epitope.

To obtain human antibody clones recognizing other epitopes present onthe cεmx-migis peptide an inhibition ELISA technique was devised whereantibodies specific for both the shared- and hidden-epitopes (A1c andB1, respectively) were used identify only the clones which may have adifferent epitope specificity. The unique clones isolated in thecεmx.migis screen were screened for those which were not inhibited byantibodies specific for the shared- and hidden-epitopes (see, forexample, FIGS. 3 and 4). Several different profiles were seen,antibodies which were inhibited by either A1c or B1, antibodies whichwere NOT inhibited by either A1c or B1 (see, for example FIG. 4, openarrows) in addition, several antibodies were identified which wereinhibited by both A1c and B1 (see, for example FIG. 4, solid arrows).About 25 clones were identified that were specific for csmx.migispeptide which were not inhibited by either A1c or B1, suggesting thatthese clones probably bound to an epitope other than the A1c or B1epitope, were selected for further screening. These selected Fab cloneswere converted to full length IgG with the exception of clone D9 whichlacks a light chain. FACS analysis demonstrated that D5 antibody (alsotermed D5 IgG) specifically binds only cells expressing membraneanchored IgE (FIGS. 5, 6 and 8) and ELISA studies demonstrated that D5specifically binds on the csmx.migis peptide and not the shared epitoperepresented by the PIBP peptide (FIG. 7A). Two additional clones, F4 andD9, were also found to bind selectively to 293 transfectants thatexpressed mIgE but not untransfected cells (FIG. 8). As shown in FIG.6B, the D5 antibody also did not bind to human B-cell lines RPMI 1788which expresses mIgM, Daikiki which expresses human mIgA, RAJI cellswhich secrete IgG and to CCRF-CEM which is a T-cell line or to SKO-007is a human B-lymphoid cell line that is reported to express mIgE but inpractice we and others have consistently found that this expression isvery weak and unstable (our unpublished data and Chen H Y, et al., IntArch Allergy Immunol., 2002, 128:315-24). FIG. 8 demonstrates that D5,F4 and D9 antibodies only bind to cells expressing membrane anchoredIgE. Thus, these antibodies represents fully human antibodies thatspecifically bind membrane anchored ε-chains and do not significantlycross react with other cell surface proteins. Further studies asdescribed below were carried out with clone D5. Clone D9 may beoptimized as a heavy chain only antibody or used as a heavy chainpartner to screen for a light chain partner with the appropriate bindingspecificity.

Materials and Methods

Peptides: Aminohexaminoic acid linker (Ahx) followed by a biotinylatedlysine (K-biot) residue were attached to the C-terminal end of eachpeptide. The different peptides are summarized in Table 2. All peptidesexcept (iii) and (iv) were dissolved in PBS, pH, 7.4. Peptides (iii) and(iv) were dissolved in 10% DMSO because they were not soluble in PBS.

TABLE 2 Peptides Used for Screening SEQ ID Peptide Sequence NO: (i)ε-migis peptide ELDVCVEEAEGEAPW-(Ahx)(K-Biot) 1 (ii) cεmx.migis peptideRADWPGPPELDVCVEEAEGEAPW-(Ahx)(K-Biot) 5 (iii) scrambled form of ε-migisGEDWCEVALEPAEVE-(Ahx)(K-Biot) 85 (iv) IgG migis peptideKSLSLSPELQLEESCAEAQDGELDG-(Ahx)(K-Biot) 86 (v) IgM migis peptideERTVDKSTEGEVSADEEGFEN-(Ahx)(K-Biot) 87 (vi) peptide fromTQLLCVEAFEGEEPW-(Ahx)(K-Biot) 3 phosphoinositide binding protein (vii)peptide from a human gene VKEEPVEEAEEEAPE-(Ahx)(K-Biot) 4 accessionnumber K1AA1227

Recombinant Proteins Three different recombinant proteins were used inthe study. These were (i) IgE that lacks any membrane tethering portion,(ii) IgE with 52 amino acids corresponding to the cεmx portion at theend of CH4 domain and (iii) IgE with 67 amino acids corresponding to thecεmx and -migis portion of mIgE at the end of the CH4 domain. The onlydifference between the protein that lacks the carboxyl terminalextension and the ones with the cεmx and cεmx.migis portion is at theend of the CH₄. IgE ends with the sequence SVNPGK. In other two proteinswith the cεmx and cεmx.migis at the end of CH4 domain, the CH₄ ends withSVNP. This difference corresponds with the difference seen betweensoluble IgE (sIgE) and mIgE. The vectors for these proteins were made bycloning the open reading frame coding for the heavy and light chainsunder CMV promoter in a mammalian expression vector designed forsecretion of protein into the culture medium. Because the three proteinswere expressed in exceeding low level they were not purified. Theirpresence in cell culture supernatant were monitored by a sandwich ELISAthat involved capturing the protein with an anti-human IgE Fc specificantibody (5 μg/ml in PBS, 7.4) and detecting with anti-human kappaantibody conjugated with HRP (data not shown). In another experimentthat was done to study the binding of these three different proteins bya cεmx.migis specific antibody (referred later on as D5) the proteinswere captured by D5 IgG (5 μg/ml in PBS, 7.4) immobilized on ELISAplates through anti-IgG-Fc antibodies and were detected with anti-humanIgE Fc specific antibody conjugated with HRP (see, FIG. 7C). Synagis®which is a therapeutic monoclonal antibody that binds to the F-proteinof respiratory syncytial virus was used as a negative control to showthat binding by D5 IgG was specific.

Phage Library: The phage library used in these studies is called theFAB-310 Fab library obtained from Dyax Corp. The library is a fullyhuman Fab library. The complexity of the library is over 10¹⁰ and hasbeen shown to be an effective source of antibodies against a widevariety of human and non-human antigens (Hoet R M et al., NatureBiotechnology 2005, 23, 344-8).

ε-migispeptide Panning: (1) In solution: At each round the phage librarywas blocked with 3% BSA in TPBS (0.1% Tween 20 in PBS, pH 7.2) for 1hour and then deselected on streptavidin coated magnetic beads for 2hours. The library was then incubated with the 8-migis peptidebiotinylated through a linker at the C-terminus. The peptide-phagecomplex (as well as free peptide) was captured on streptavidin coatedmagnetic beads. The beads were then washed 7 times each with TPBS andPBS, each wash being of 2′ duration. Following the washes the phage waseluted using 100 mM Triethylamine in water for 15 minutes. The elutedphage was immediately neutralized with 0.5 M Tris, pH 8, titered andamplified by infecting E. coli for subsequent round of panning. Peptideconcentrations of 2.0 μg/ml (˜1 μM) were used for rounds 1 and 2 and0.20 μg/ml (˜0.1 μM) was used for round 3. (2) Immobilized: As describedfor the solution panning the phage library was blocked with 3% BSA inTPBS (0.1% Tween 20 in PBS, pH 7.2) and then deselected on Neutravidincoated immunotubes coated with 1 ml of 2 mg/ml of Neutravidin. In aseparate tube the biotinylated ε-migis peptide was captured on a 5 μg/mlcoated neutravidin surface and the deselected library was incubated onthe captured peptide bed. The bed was washed 15 times each with TPBS andPBS and the phage were eluted with 100 mM Triethylamine in water for15′. As described above the eluted phage was immediately neutralized,titered and amplified by infecting E. coli for subsequent round ofpanning. Peptide concentrations of 25 μg/ml (˜13 μM) were used forrounds 1 and 2 and 2.5 μg/ml (˜1.3 μM) was used for round 3.Approximately 500 clones were screened by ELISA for those that boundonly to the ε-migis peptide and not to a scrambled peptide. There were atotal of 51 (10%) ELISA positive clones representing 18 unique Fabs.These 18 clones were batch converted to IgG. Of the full IgG clonesrecovered 12 specifically bound 8-migis but not to a scrambled peptide.Only one clone, A1c, showed the ability to bind mIgE expressing cellsbut was subsequently found to bind cells even in the absence of mIgE andlater determined to bind the shared epitope (see, FIG. 7A).

cεmx.migis peptide Panning: Panning was performed essentially asdescribed above for e-migis peptide panning (1) In solution: the blockedlibrary was deselected on streptavidin coated magnetic beads thenincubated with the csmx.migis peptide biotinylated through a linker atthe C-terminus. The peptide-phage complex (as well as free peptide) wascaptured on streptavidin coated magnetic beads. The beads were thenwashed and the phage eluted. Peptide concentrations of 2.0 μg/ml wereused for rounds 1 and 2 and 0.25 μg/ml was used for round 3. (2)Immobilized: the blocked library was deselected on Neutravidin coatedimmunotubes. The biotinylated cεmx.migis peptide was captured on aneutravidin surface and the deselected library was incubated on thecaptured peptide bed. The bed was washed and eluted. Peptideconcentrations of 25 μg/ml were used for rounds 1 and 2 and 2.5 μg/mlwas used for round 3. Approximately 2500 isolated clones were thenscreened by ELISA for binding to the cεmx.migis peptide, 364 werepositive. The positive clones were then screened for binding to both thecsmx.migis and the 6-migis peptides. 256 clones bound to both cεmx.migisand the ε-migis peptides while 108 preferentially bound to cεmx.migis.Of 190 clones sequence analyzed, 120 were unique. 8 randomly pickedclones were initially converted to IgGs. However, none specificallybound to cell expressing mIgE. The 120 unique clones from panning on thecεmx-migis peptide were screened by inhibition ELISA for those that werenot strongly inhibited by A1c. The results from some representativeclones screened against A1c are shown in FIG. 3. A total of 66 cloneswere selected. These clones were consolidated and screened by inhibitionELISA for those clones that were not inhibited by A1c or B1. FIG. 4shows the results from some representative clones screened against bothA1c and B1.

ELISA Screening: For screening studies phage particles from singlebacterial colonies were rescued in 96-well formats as described inChowdhury et al. (Mol. Immunol. 1997 January; 34(1):9-20.). Thebacterial culture was then cooled down to 4° C. and the cells wereremoved by centrifugation at 3000-5000×g for 15′ at 4° C. Thesupernatant containing recombinant phage particles were used forscreening assays. Biotinylated cεmx-migs peptide was immobilized onNeutravidin coated ELISA plates that had been blocked with 3% BSA inTPBS. After blocking the wells bacterial culture supernatant containingindividual phage clones were added to the ELISA wells. After incubationfor 60′ at ambient temperature the wells were washed and the bound phagewere detected with anti-M13 antibody conjugated to HRP. ELISA plateswere developed with TMB substrate solution from Pierce. The reaction wasstopped with 2 N sulphuric acid and the intensity of the color producedwas measured at 450 nm.

Inhibition ELISA Screening: Biotinylated cεmx-migs peptide wasimmobilized on Neutravidin coated ELISA plates as described above.Bacterial cultures containing individual phage clones isolated bypeptide panning were added to separate ELISA wells along with anirrelevant IgG isotype (specific towards the F-protein of RespiratorySyncytial Virus) control or A1c (anti-shared epitope antibody) or B1(anti-hidden epitope antibody). After incubation for 60′ at ambienttemperature the wells were washed and the bound phage clones weredetected using an anti-M13 antibody coupled to HRP as described above.Some representative ELISA data from the inhibition screening is shown inFIGS. 3 and 4. Several clones, namely A8, CP3-B1, C3, C11, D2, D5, D8,D9, F4 and E6 were selected for conversion to IgG and analyzed forbinding to membrane IgE transfected cells (see below).

Conversion and Expression of Full Length IgGs: The Fab gene in the phagedisplay vector consist of the entire light chain separated from the Fdfragment by a DNA piece that represent a bacterial ribosome entry site(RBS). To convert a Fab fragment selected from the phage library intofull IgG the Fab cassette was isolated by using unique restrictionenzymes, ApaLI and NheI and ligated into a mammalian expression vectorsuch that the Fd fragment is ligated in frame with the rest of theconstant domains of the heavy chain. After this the bacterial RBS wasremoved by two other restriction sites and replaced by an IRES sequencethat works for mammalian expression systems. The final vector is thusmonocistronic where the light and heavy chain are transcribed into onemRNA but translated as two different chains.

Conversion of the Fabs to full IgGs was followed by the expression ofthe IgGs in 293 cells by transient transfection. 293 cells weretransfected (using Lipofectamine 2000 from Invitrogen following themanufacturer's instruction) with the mammalian expression vectors codingfor the IgG and cultured for 72 hours in DMEM containing ultra low IgGcontaining 10% FBS from Invitrogen. The IgG concentration in theconditioned media were estimated by a sandwich ELISA in which anti-humankappa antibody from Sigma was used to capture the human IgG from theconditioned culture media and anti-human IgG Fc antibody conjugated toHRP was used to detect the captured IgG. The amount of IgG present inthe conditioned media was estimated by using purified human IgG1-k as acontrol antigen to generate a standard curve in an experiment run inparallel. After normalizing for the IgG concentration the conditionedsupernatants were screened by ELISA for peptide binding and then bindingto mIgE expressing cells in a FACS based assay.

Cell Line Generation: Nucleoporation was used for generating transfectedcell lines. 293 cells were co-transfected with (i) a linearizedbi-cistronic mammalian expression vector coding for the mIgE heavy chainand a light chain that binds to the EphA2 antigen or with another mIgEheavy and light chain pair that binds to the F-protein of RespiratorySyncytial Virus (RSV) and (ii) a linearized bi-cistronic mammalianexpression vector that coded for CD79a (Ig α) and CD79b (Ig β) that areknown to associate with membrane immunoglobulins to form the B-cellreceptor complex (BCR). 24 hours after transfection cells were seeded at500 cells/well of 96 well plates and subjected to double selection by500 μg/ml neomycin (for the mIgE expressing plasmid) and 100 μg/mlhygromycin (for the CD79a and CD79b expressing plasmid) in Dulbecco'sModified Eagle's Medium (DMEM) (from Invitrogen, Carlsbad, Calif.).After 2-3 weeks colonies started to emerge. These were expanded andtested for expression of mIgE, CD79a and CD79b. The population with goodexpression of all three antigens was sorted by three color FACS intosingle cell/well giving rise to several clones with consistentexpression of mIgE, CD79a and CD79b. These clones were then furthersub-cloned by limited dilution cloning at 0.2 cell/well to ensuremonoclonality. FIG. 17 is a bar graph of the FACS analysis of cellsurface staining with anti-hu IgE, anti-Igα, anti-Igβ and a secondaryantibody control demonstrating that clones 1, 2 and 5 stain for allthree cell surface markers.

FACS Analysis: For FACS experiment all steps were carried out at 4° C.The 8 clones identified by competition ELISA were tested for theirability to bind mIgE. Briefly, cells transiently expressing mIgE(293-mIgE) were stained with anti-human κ, anti-human λ, anti-humanIgG-Fc and antibodies derived from each of the clones described above(AS, CP3-B1, C3, C11, D2, D5, D8, and E6) as well as, A1c and B1 (shownas CP1-B1). Cells were then analyzed by FACS (see FIG. 5).

Cells expressing mIgE (293-mIgE), IgA (Daikiki), IgM (RPMI 1788) or noimmunoglobulin (293 and CCRF-CEM) were stained with, secondary antibodyalone (goat anti-mouse (GtαMu) or Guinea pig anti-human (GαHu)) orprimary (mouse anti-human IgE (MuαHuIgE) or D5) plus secondary or leftunstained. Cells were then analyzed by FACS (see FIG. 6). Because theexperiment was done with transiently transfected 293 cells both thepercentage of cells showing staining and the change in mean fluorescencewas plotted in FIG. 6A.

Similar experiments were performed using 2×10⁴ 293 or 293 stablytransfected with mIgE, CD79a and CD79b or other human B-cell lines suchas RAJI, RPMI 1740, Daikiki or the T-cell line CCRF-CEM. The cells werefirst blocked with 2% BSA in PBS (blocker). They were then exposed tothe conditioned media containing 2-5 μg human IgG in 200 μl blocker for45′. Cells were washed three times using 5 ml blocker and centrifugationfor 5′ at 400 g. The cells were then stained with anti-human IgG1 Fcspecific antibody labeled with Alexar 488 and analyzed in a Guava. IgGsof clones that showed binding were then studied further by doing theFACS staining both in the absence and presence of the migis or ascrambled peptide (20 ug/ml) (data not shown).

6.2 Example 2 Binding Characteristics of D5, a Human Anti-cεmx.migisAntibody

To further characterize the binding specificity of the human anticεmx.migis antibody D5, a recombinant IgE antibody (rIgE), was generatedand expressed in 293 cells. Several variants of soluble rIgE in whichthe cεmx region (52 amino acid residues) or the cεmx-migis region (67amino acid residues) were fused to the CH4 end (designated rIgE.cεmx andrIgE.cεmx.migis, respectively) were also generated and expressed. ELISAanalysis demonstrated that the D5 antibody binds only rIgE.cεmx.migisand not IgE or IgE.cεmx indicating that D5 does not significantly bindto the cεmx region alone. This suggests that the cεmx.migis specific D5antibody does not bind to soluble IgE and binds IgE only when it hasboth the cεmx and the migis peptide at its C-terminus. These data alsosuggest that a portion of the ε-migis peptide is involved in forming theepitope recognized by D5 (FIG. 7B).

A second series of recombinant immunoglobulin molecules were alsogenerated in which the cεix region or the cεmx-migis region were fusedto the CH₃ end of an IgG (designated rIgG.cεmx and rIgG.cεmx.migis,respectively). ELISA analysis again demonstrated that the D5 antibodyonly binds rIgG.cεmx.migis (FIG. 7C). This data indicates that theconfirmation of cεmx.migis region may be similar in the context of bothIgE and IgG constant regions.

To further define the epitope recognized by D5 BIAcore analysis ofbinding to cεmx.migis and ε-migis peptides was performed. As shown inFIG. 9, D5 does not bind to the ε-migis peptide alone but rather bindsto an epitope only present in the cεmx.migis peptide. This data confirmsthe ELISA studies described above indicating that both the cεmx and theε-migis amino acid residues are involved in forming the epitoperecognized by D5.

Materials and Methods

Generation of rIgE and rIgE variants: the variable region of the heavychain of an irrelevant antibody was used to generate a recombinant IgE(rIgE). The entire cεmx region (52 residues total) or the combinedcεmx.migis region (67 residues) was fused to the CH4 end of the rIgEconstruct to generate rIgE.cεmx and rIgE.cεmx.migis, respectively. Allthree constructs were transfected into 293 cells for expression and therelative level of protein expressed was determined by a sandwich ELISA.Briefly, the various rIgE constructs were captured with an anti-lightchain antibody and detected with an anti-IgE Fc antibody conjugated toHRP.

rIgE Binding ELISA Assay: D5 or the isotype control antibody (cont.)were captured by an anti-IgG Fc antibody bound to an ELISA plate.Conditioned media from cells expressing the various rIgE constructs wereadded to the ELISA wells and binding was detected using an anti-IgE Fcantibody conjugated to HRP.

BIAcore Analysis: the interaction of D5 with streptavidin-capturedcεmx.migis and ε-migis peptides was monitored by surface plasmonresonance detection using a BIAcore 3000 instrument (PharmaciaBiosensor, Uppsala, Sweden). Briefly, the peptides were captured on astreptavidin coated chip and the D5 antibody was passed over thesurface.

6.3 Example 3 Biological Characteristics of D5, a Human Anti-cεmx.migisAntibody

D5 was tested in an ADCC assay to demonstrate that a human antibodyagainst cεmx.migis was useful for depleting cell expressing membranebound IgE. D5 was seen to mediate ADCC activity only against 293 cellthat were transfected to express membrane bound IgE (FIGS. 10 and 11).The ability to mediate ADCC activity could be specifically inhibited bythe addition of the cεmx.migis peptide (FIG. 11).

A valiant of the D5 antibody was generated having an aspartate atposition 239, a leucine at position 330 and a glutamate at position 332in the Fc region of the antibody (the numbering system is that of the EUindex as set forth in Kabat et al., 1991, NIH Publication 91-3242,National Technical Information Service, Springfield, Va.), this variantwas designated “D53M”. The presence of these amino acid residues atthese positions in the Fc domain of IgG has been shown to enhance ADCCactivity. As shown in FIGS. 10 and 11, an enhancement in ADCC activityis seen for the D53M Fc variant antibody. As seen for the D5 antibody,the ability of the D53M antibody to mediate ADCC is inhibited by thecεrmx.migis peptide (FIG. 11).

Materials and Methods

Generation of D53MFc variant: To generate the D53M variant the variableregion of the D5 heavy chain was fused to an Fc region variant havingthe following non-wild type amino acid resides, 239D, 330L and 332E.

Preparation of Peripheral Blood Mononuclear Cells (PBMCs): Human Bloodsamples were collected from several individual healthy volunteers usingheparinized syringes, diluted with twice the volume of PBS buffer,layered onto a Lymphoprep gradient (ICN, Irvine, Calif.), andcentrifuged at 400 g for 30 minutes at room temperature. Peripheralblood mononuclear cells (PBMCs) were harvested from the interface,washed 3 times with PBS.

ADCC: Antibody-dependent cell cytotoxicity (ADCC) was assayed in anon-radioactive lactate dehydrogenase (LDH) release assay (PromegaCorporation, Madison, Wis.). Briefly, 293 or 293-mIgE target cells weredistributed into 96-well plates and pre-incubated with serial dilutionof antibodies (50 μL) for 20 min at 37° C. Human effector cells (100 μl)were then added at ratios from 25:1 to 50:1. Human effector cells wereperipheral blood mononuclear cells (PBMC) purified from healthy donorsusing Lymphocyte Separation Medium (MP Biomedicals, Irvine, Calif.).After incubation, plates were centrifuged, and cell death was analyzedby measuring the release of LDH into the cell supernatant with a30-minute coupled enzymatic assay. The percentage of specific lysis wascalculated according to the formula: % specificlysis=100×(E_(X)−E_(spon)−T_(spon))/(T_(max)−T_(spon)), where E_(X)represents the release from experimental wells, E_(spon) is thespontaneous release of effector cells alone, T_(spon) is spontaneousrelease of target cells alone, and T_(max) is the maximum release fromlysed target cells.

Additional assays were performed using 293 cells and 293 cellsexpressing mIgE and CD79a and CD79b (FIG. 10C). The 293 cells and 293cells expressing mIgE and CD79a and CD79b were harvested using celldissociation buffer (from Invitrogen) and re-suspended in RPMI 1640supplemented with 5% FBS (assay buffer) at a density of 2×10⁵ cells/ml.These were then added to a 96-well round bottom tissue culture plate (BDBiosciences, Bedford, Mass.) at 50 μl/well along with variousconcentrations of antibody at 50 μl/well in assay buffer (see above) andpre-incubated at 37° C. for 30 minutes. PBMCs were resuspended at 5×10⁶cells/1 ml (for an Effector (E):Target (T) ratio of 50:1) and 2.5×10⁶/ml(for an E:T ratio of 25:1) in assay buffer (see above) and added at 100μl/well to the assay plate. 25 μl/well of 9% Triton X-100 (Promega,Madison, Wis.) was added as a control for complete lysis. The plateswere centrifuged at 300 g for 3 minutes and incubation at 37° C. wascontinued for 4 hours. Plates were then centrifuged at 300 g for 10minutes and 50 μl of supernatant from each well was transferred toMaxiSorp 96-well plates (BD Biosciences, Bedford, Mass.). 50 μl ofreconstituted substrate mix (CytoTox 96 Non-Radioactive CytotoxicityAssay kit, Promega, Madison, Wis.) was then added to all wells andincubated in the dark at room temperature for 30 minutes. 50 μl of stopsolution (Promega, Madison, Wis.) was added to each well and lactatedehydrogenase (LDH) release was quantified by measuring the absorbanceat 490 nm. % cytotoxicity was calculated as described above.

Whereas, particular embodiments of the invention have been describedabove for purposes of description, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety and for all purposesto the same extent as if each individual publication or patent or patentapplication was specifically and individually indicated to beincorporated by reference in its entirety for all purposes. In addition,U.S. Provisional Patent Application No. 60/721,525 filed Sep. 29, 2005is incorporated by reference in its entirety for all purposes.

1. An isolated antibody or antibody fragment thereof which specificallybinds the peptide sequence of SEQ ID NO:5, wherein said antibody orantibody fragment thereof does not bind membrane-anchoredimmunoglobulins other than membrane-anchored immunoglobulin E (mIgE). 2.The isolated antibody or antibody fragment of claim 1, wherein saidantibody or antibody fragment does not bind at least one polypeptideselected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ IDNO:4 and SEQ ID NO:6.
 3. (canceled)
 4. The isolated antibody or antibodyfragment claim 1, wherein said antibody or antibody fragment does notbind the same epitope as antibodies or antibody fragments comprising thevariable regions of A1c (encoded by SEQ ID NOS: 50 and 51) and B1(encoded by SEQ ID NOS: 60 and 61).
 5. The isolated antibody or antibodyfragment of claim 1, wherein the binding is not inhibited by antibodiesor antibody fragments comprising the variable regions of A1c (encoded bySEQ ID NOS: 50 and 51) and B1 (encoded by SEQ ID NOS: 60 and 61). 6-8.(canceled)
 9. The isolated antibody or antibody fragment thereof ofclaim 1, wherein said antibody or fragment thereof depletes B cells orplasma cells expressing mIgE.
 10. The isolated antibody or antibodyfragment of claim 9, wherein said antibody or antibody fragment depletessaid B cells or plasma cells through ADCC. 11-14. (canceled)
 15. Theisolated antibody or antibody fragment of claim 1, said antibody orantibody fragment comprising: a. a light chain variable region havingthe 3 CDRs of the light chain of the antibody D5 encoded by SEQ ID NO:7, and a heavy chain variable region; b. a heavy chain variable regionhaving the 3 CDRs of the light chain of the antibody D5 encoded by SEQID NO: 8, and a light chain variable region; c. a light chain variableregion having the 3 CDRs of the light chain of the antibody D5 encodedby SEQ ID NO: 7, and a heavy chain variable region having the 3 CDRs ofthe light chain of the antibody D5 encoded by SEQ ID NO: 8; d. a lightchain variable region having the 3 CDRs of the light chain of theantibody F4 encoded by SEQ ID NO: 70, and a heavy chain variable region;e. a heavy chain variable region having the 3 CDRs of the light chain ofthe antibody F4 encoded by SEQ ID NO: 71, and a light chain variableregion; f. a light chain variable region having the 3 CDRs of the lightchain of the antibody F4 encoded by SEQ ID NO: 70, and a heavy chainvariable region having the 3 CDRs of the light chain of the antibody F4encoded by SEQ ID NO: 71; g. a heavy chain variable region having the 3CDRs of the light chain of the antibody D9 encoded by SEQ ID NO: 80; orh. a heavy chain variable region having the 3 CDRs of the light chain ofthe antibody D9 encoded by SEQ ID NO: 80, and a light chain variableregion. 16-20. (canceled)
 21. The isolated antibody or antibody fragmentof claim 1, wherein the antibody comprises a variable light chaincomprising the amino acid sequence of SEQ ID NO:9, or SEQ ID NO:
 72. 22.The isolated antibody or antibody fragment of claim 1, wherein theantibody comprises a variable heavy chain comprising the amino acidsequence of SEQ ID NO: 10, SEQ ID NO: 73 or SEQ ID NO:
 81. 23-25.(canceled)
 26. An isolated nucleic acid sequence coding for an aminoacid sequence selected from the group consisting of SEQ ID NO:9, 10, 72,73 and
 81. 27. An isolated cell comprising at least one nucleic acidsequence selected from the group consisting of SEQ ID NO: 7, 8, 70, 71and
 80. 28-38. (canceled)
 39. The isolated antibody or antibody fragmentof claim 1, wherein said antibody or antibody fragment is a humanized orhuman antibody or antibody fragment.
 40. (canceled)
 41. The isolatedantibody or antibody fragment of claim 1, wherein said antibody orantibody fragment is selected from the group consisting of: a. an scFv;b. a Fab fragment; c. an Fab′ fragment; d. an F(ab)₂; e. an Fv; f. adisulfide linked Fv; and g. a bi-specific antibody.
 42. A pharmaceuticalcomposition comprising the isolated antibody or antibody fragment ofclaim
 1. 43. A method of producing the isolated antibody or antibodyfragment of claim 1 comprising: a. screening an antibody library beforeor after selection for antibodies that bind SEQ ID NO:5 and bind with atleast 5-fold less affinity to SEQ ID NO: 1 and SEQ ID NO:6 b. isolatingat least one antibody from (a). 44-46. (canceled)
 47. A method ofpreventing, ameliorating, or treating an IgE-mediated disease in a humancomprising administering to an individual in need of such prevention,amelioration, or treatment an effective amount of the isolated antibodyor antibody fragment of claim
 1. 48-49. (canceled)
 50. A method ofproducing an antibody that does not bind to a predominant epitopecomprising: a. screening an antibody library before or after selectionfor antibodies which bind to a polypeptide comprising a predominantepitope for antibodies which are not inhibited by an antibodyrecognizing the predominant epitope present on said polypeptide; and b.isolating at least one antibody from (a).
 51. The method of claim 50,wherein the screening step takes place after selection for antibodieswhich bind to a polypeptide comprising a predominant epitope.
 52. Amethod for producing antibodies that specifically bind a migis epitopewhich is not a predominant epitope and which is not hidden on amembrane-anchored immunoglobulin comprising: a. isolating from anantibody library those clones which bind to a polypeptide comprising themigis epitope; b. screening the clones isolated from step (a) for thosewhich are not inhibited by an antibody recognizing the predominantepitope present on said polypeptide comprising the migis epitope; c.screening the clones which are not inhibited in step (b) for those whichspecifically bind cells having the membrane anchored immunoglobulin; andd. isolating at least one antibody from (c).
 53. The method of claim 52wherein step (c) further comprises screening for those clones which donot bind cells not having the membrane anchored immunoglobulin.